Fusion protein for secretory protein expression

ABSTRACT

There is provided a fusion protein suitable for secretion of more than one polypeptide(s) of interest (POI) comprising a signal peptide, a POI, a passenger domain comprising a beta stem domain from an autotransporter protein, and a translocator domain from an autotransporter protein, wherein the beta stem-forming sequence of the passenger domain is essentially intact and the POI(s) is/are fused to the beta stem domain.

TECHNICAL FIELD

The present invention relates generally to a novel fusion protein and amethod for secretory protein expression.

BACKGROUND ART

Secretory protein expression is the expression of a protein in a hostcell, where the protein is exported to the cell membrane and is eithersolubly released into the medium or remains attached to the cellmembrane. Secretory protein expression is mediated by a signal peptideat the N-terminus of the protein which directs the polypeptide to themembrane.

Usually, recombinant proteins that are produced in prokaryotic hostssuch as E. coli are produced intracellularly. When the protein isrecovered in such a procedure, the cells have to be lysed which leads tocontamination of the recombinant protein with cellular content. Theprotein then has to be recovered from whole cell extracts in multi-steppurification procedures, which is time consuming and results in pooryields.

Secretion of recombinant proteins into the medium is a better strategybecause purification of proteins from spent medium is easier and morecompatible with continuous culturing. However, the present systems donot have efficient yields.

Secretory protein expression where the protein remains attached to thecell surface has other uses. Examples of use for this type of proteinexpression include live-vaccine development, epitope mapping, biosorbentand biosensor development and the high throughput screening of proteinand peptide libraries for drug discovery.

In both surface display and secretion, recombinant proteins face thechallenge of translocation across the complex E. coli cell envelope thatconsists of two lipid membranes (the inner and outer membrane) with agel-like compartment, the periplasm, in between. This has been shown tobe very difficult and the methods previously used have had low efficacy.

Autotranporters are large proteins that are secreted by Gram-negativebacteria, such as E. coli. The autotransporter system is simple in thesense that the autotransporter, as implied by its name, is suggested tocarry all information for translocation across the periplasm and outermembrane within the protein itself. However, the mechanism wherebyautotransporters are secreted is still not completely understood.

Autotransporters are synthesized as large precursor proteins thatcontain three main domains: (i) an N-terminal signal peptide thattargets the protein to the Sec translocon and initiates transfer acrossthe inner membrane, (ii) a passenger domain which comprises the “cargo”protein that is to be secreted and (iii) a C-terminal pore-formingdomain (translocator domain) comprising a beta barrel structure thatintegrates into the outer membrane and plays a crucial but unclear rolein translocation of the passenger domain across the outer membrane intoextracellular space.

After translocation, the passenger domain is cleaved from thetranslocator domain and is released into the extracellular environment.In some cases, the passenger domain remains non-covalently attached tothe cell surface. Cleavage can be achieved by the action of an(external) protease on a protease motif situated between thetranslocator domain and the passenger domain. Alternatively, cleavagetakes place through an intramolecular autocatalytic event at a specificsite between the translocator domain and the passenger domain.

The passenger domain of an autotransporter comprises a beta stemstructure and side domains. The beta stem is an elongated structureformed by an extended beta helix. The C-terminus of the passenger domaincomprises an autochaperone domain which has been implicated in bothpassenger folding and translocation across the outer membrane.

Hbp is an autotransporter protein that belongs to the subfamily ofserine protease autotransporters of Enterobacteriaceae (SPATEs). Thecrystal structure of the passenger domain of Hbp has recently beendetermined (Otto et al. 2005 J Biol Chem 280(17): 17339-45), and isshown as FIG. 11A. The structure shows that the polypeptide forms a longright-handed beta-helical structure (“beta stem”). The passenger domainof the Hbp comprises two larger side domains, domain d1 and domain d2,of which d1 comprises the serine proteinase activity of the protein andd2 has an unknown function. There are also three smaller side domains,domain 3 (d3), domain 4 (d4) and domain 5 (d5).

Similar beta stem domains have been shown also for otherautotransporters such as pertactin (Emsley et al 1996 Nature 381: 90-92)and IgA protease (Johnson et al 2009 J Mol Biol 389(3): 559-74).

There have been previous attempts in using autotransporters forsecretory protein expression in E. coli, mostly using variants of theNeisserial IgA protease (Pyo et al 2009 Vaccine 27 2030-2036) and theendogenous E. coli autotransporter AIDA-1 (Van Gerven et al 2009Microbiology 155:468-476) that were engineered for surface displaypurposes.

Efforts using IgA protease and AIDA-1 for secretion of recombinantproteins used constructs which resulted in poor yields of secreted andsurface exposed protein (Pyo et al 2009 Vaccine 27 2030-2036; Van Gervenet al 2009 Microbiology 155:468-476). In the majority of such studiesthe complete, or almost complete, endogenous passenger domain wasreplaced by the recombinant protein.

So far, autotransporters have mainly been used as a display platformrather than for secretion of heterologous proteins in soluble form,where the protein is secreted into the medium.

IgA protease requires an accessory protease for processing whereasAIDA-1 remains non-covalently attached to the outer membrane aftercleavage. Thus, these autotransporters can only be used for surfacepresentation of epitopes and proteins.

Efficient display and secretion of calmodulin fused the passenger of Hbphas previously been shown (Jong et al 2007 Molecular Microbiology63:1524-1536). In order to minimize perturbation of the native β-stem ofthe passenger, calmodulin replaced domain 2 of the Hbp passenger.

For certain applications the possibility to secrete or display more thanone protein of interest (POI) from/on the cell surface is very useful.Such applications include vaccines, for example in which two or moreepitopes are displayed on the same cell surface, enzyme display, inwhich more than one enzyme is displayed on the cell surface in order tocarry out a range of catalytical reactions in a series of steps,exposure of peptide libraries and inhibitor screening.

For multivalent vaccines it is particularly useful to have a systemwherein one population of host cells can express and display or secretemultiple antigens, rather than having a mixture of cell populations,each displaying or secreting only one of the antigens. Having only onecell population displaying or secreting multiple antigens has theadvantage of easier production and better control of the vaccinecontent.

In conclusion, there is a need for improved secretory expressionssystems for the display of heterologous proteins as well as secretion ofheterologous proteins in soluble form into the culture medium. There isalso a need for a system and a method that enable secretory proteinexpression of more than one protein of interest on the cell surface of ahost cell or secretion of more than one protein into the culture medium.

OBJECT OF THE INVENTION

An object of the present invention is to provide efficient secretion ofa polypeptide of interest (POI) from a host cell.

A second object of the invention is to provide efficient display of aPOI on the surface of a host cell.

A third object of the invention is to provide efficient solublesecretion of a POI into the medium in which a host cell is cultured.

Yet another object of the invention is to provide a scaffold forefficient secretion, i.e. display or soluble secretion, of more than onePOI.

SUMMARY OF THE INVENTION

In a first aspect of the invention there is provided a host cell capableof expressing more than one, such as at least two, POI:s (proteins ofinterest). The POI:s are comprised in a fusion protein that alsocomprises a passenger domain comprising a beta stem domain from anautotransporter protein, a translocator domain from an autotransporterprotein, and a signal peptide that is able to target the fusion proteinto the inner membrane of Gram negative bacteria. The beta stem formingsequence of the passenger domain is essentially intact and the POI:s arefused to the passenger domain.

This host cell for secretory protein expression has several advantages,including but not limited to more efficient secretion of more than onePOI, compared to other systems. Also, when the goal is to display thePOI, the beta stem domain will enable a more efficient display as thePOI:s will be further away from the cell surface and be more stable.

In one embodiment of the host cell of the present invention, the nativeform of the passenger domain of the autotransporter comprises at leastone side domain that protrudes from the beta stem domain. The POI:s maythen be inserted into, replace or partly replace such side domain.

In another embodiment the native form of the passenger domain of theautotransporter comprises at least two side domains. Each POI may thenbe inserted into, replace or partly replace a separate such side domain,or the POI:s may be inserted into, replace or partly replace the sameside domain.

The POI:s may also be fused to an independent passenger domain,translocator domain and signal peptide from an autotransporter.

In a second aspect of the invention there is provided a fusion proteincomprising more than one, such as at least two, POI:s (proteins ofinterest), a passenger domain comprising a beta stem domain from anautotransporter protein, a translocator domain from an autotransporterprotein, and optionally, a signal peptide that targets the fusionprotein to the inner membrane of a Gram negative bacteria. The beta stemforming sequence of the passenger domain is essentially intact and thePOI:s are fused to the passenger domain.

The passenger domain of the fusion protein may in its native formcomprise at least one side domain protruding from the beta stem domain,and the POI:s may be inserted onto, replace or partly replace such sidedomain. The passenger domain of the fusion protein may also in itsnative form comprise at least two side domains, and each POI may beinserted into, replace or partly replace independent domains of suchside domains. Alternatively the POI:s may be inserted into, replace orpartly replace the same side domain.

In another aspect of the invention there is provided a nucleic acidarranged for expression of a fusion protein. In one embodiment thenucleic acid comprises, in frame, sequence encoding a signal peptide ofthe fusion protein, that is able to target the fusion protein to theinner membrane of Gram negative bacteria, sequence encoding a passengerdomain of the fusion protein, that comprises a beta stem domain from anautotransporter protein, and sequence encoding a translocator domain ofthe fusion protein, that derives from an autotransporter protein. Thesequence encoding the passenger domain comprises at least two stretchesof cloning site sequence that allow in-frame cloning of at least two DNAsequences that encode POI:s (proteins of interest). The cloning sitesequences are arranged such that the encoded beta stem forming proteinsequence of the passenger domain is essentially intact. It is alsopossible to insert POI:s into an autotransporter by merely fusing twopieces of DNA, e.g. by PCR, without using cloning sites thereby creatinga fusion protein.

The sequence encoding the passenger domain of the autotransporter may inits native form comprise at least two stretches of sequence encodingside domains protruding from the beta stem domain. The at least twostretches of cloning site sequence may then be inserted into, replace orpartly replace separate of such stretches encoding side domains.

In another embodiment the nucleic acid comprises, in frame, sequenceencoding a signal peptide of the fusion protein, that is able to targetthe fusion protein to the inner membrane of Gram negative bacteria,sequence encoding a passenger domain of the fusion protein, thatcomprises a beta stem domain from an autotransporter protein, sequenceencoding a translocator domain of tha fusion protein, that derives froman autotransporter protein, and sequences encoding at least two POI:s ofthe fusion protein. The sequences encoding the POI:s are fused to thesequence encoding the passenger domain and are arranged such that theencoded beta stem forming protein sequence of the passenger domain isessentially intact.

The sequence encoding the passenger domain of the autotransporter in itsnative form may comprise at least two stretches of sequence encodingside domains protruding from the beta stem domain. Each of the at leasttwo sequences encoding POI:s may then be inserted into, replace orpartly replace each of the stretches encoding side domains.

The host cell, fusion protein or nucleic acid may be arranged such thatthe fusion protein, when expressed, is secreted from the cell surface.For instance, the fusion protein may comprise a cleavage site thatallows the fusion protein to be cleaved and secreted from a host cellexpressing the fusion protein. And the nucleic acid encoding the fusionprotein may encode such a cleavage site.

Alternatively, the host cell, fusion protein or nucleic acid may bearranged such that the fusion protein, when expressed, is displayed atthe cell surface. For instance the fusion protein may comprise no suchcleavage site or may comprise a disrupted cleavage site. Similarly thenucleic acid encoding the fusion protein then encodes no such a cleavagesite or encodes a disrupted cleavage site. Alternatively, the fusionprotein and nucleic acid may comprise a cleave site and the resultingfusion protein be cleaved, but remains non-covalently attached to, andthus displayed at, the cell surface.

The passenger domain and the translocator domain may be derived from aSPATE (serine protease autotransporters of Enterobacteriaceae) protein,such as Hemoglobin-binding protease (Hbp), extracellular serine protease(EspC) or temperature-sensitive hemagglutinin (Tsh) from Escherichiacoli.

In one aspect of the invention there is provided a vector comprising anucleic acid of the invention.

In another aspect of the invention there is provided a host cellcomprising a nucleic acid or a vector of the invention.

In one embodiment the host cell of the invention is a Gram negativebacterium, which may be selected from the family of Enterobacteriaceae,such as Escherichia coli, Salmonella spp., Vibrio spp., Shigella spp.,Pseudomonads spp., Burkholderia spp. or Bordetella spp.

In one aspect there is provided an outer membrane vesicle displaying afusion protein according to the invention. In another aspect there isprovided a bacterial ghost displaying a fusion protein according to theinvention.

In one aspect there is provided a method for secretory proteinexpression of a fusion protein, comprising the steps of providing a hostcell according to the invention and inducing expression of the fusionprotein.

In one embodiment the method comprising the additional step ofinhibiting a periplasmic enzyme, such as DegP, with protease activity inthe host cell. DegP may for example be inhibited by a mutation in itscatalytic site.

In another embodiment the method comprises the additional step of downregulating at least one enzyme, such as DsbA or DsbB, that catalyzes theformation of disulphide bonds in proteins in the periplasmic space ofthe host cell.

The method may provide secretion of the fusion protein in a solublemanner. Alternatively it may provide display of the fusion protein onthe cell surface.

In one embodiment the method comprises the additional step of inducingshedding of vesicles from the outer membrane of the host cell, being aGram negative bacterium, thus forming outer membrane vesicles displayingthe fusion protein on their surface.

In another embodiment the method comprises the additional step of lysingthe Gram negative bacterium, thus forming bacterial ghosts displayingthe fusion protein on their surface. The lysing may be made by use ofthe lethal lysis gene E from bacteriophage PhiX174.

In one embodiment at least one of the POI:s may comprise an antigen, forexample from an infectious organism. The antigen is for example anantigen from Mycobacterium tuberculosis, such as ESAT-6, Ag85B, Rv2660c,TB10.4 and TB10.3, or a protein that is similar to those proteins.

In one aspect there is provided a vaccine comprising a host cell, afusion protein, an outer membrane vesicle or a bacterial ghost accordingto the invention.

BRIEF DESCRIPTION OF FIGURES

The invention is now described, by way of example, with reference to theaccompanying figures, in which:

FIG. 1-10 show plasmid maps of plasmids used in the examples.

FIG. 11 A-D show figures of the structure of the passenger domain of Hbpwhere certain domains are indicated.

FIG. 11 E shows various constructs of fusion proteins used in examples1-15.

FIG. 12-33 show experimental data from examples 1-19. For details, seethe example section.

FIG. 34 shows a map of a plasmid used in the examples.

DEFINITIONS

As used herein, the following definitions are supplied in order tofacilitate the understanding of the present invention.

An “autotransporter” is a protein that belongs to the pfamautotransporter family (‘Autotransporter’ PF03797) and that also isknown or predicted to form a beta stem motif. The BETAWRAPPRO method forsequence analysis can be used to predict if the passenger domain of anautotransporter will form a beta stem motif (Junker et al 2006 Proc NatlAcad Sci USA 103(13): 4918-23).

A “polypeptide of interest” (POI) is a polypeptide that the user of theinvention wants a host cell to secrete in soluble form into the mediumor to display on the cell surface, or both. Typically, the POI is aprotein that the user studies or wants to be expressed, whereas theother parts of the fusion protein assist in the secretion process.Typically, the POI is also heterologous to the autotransporter domainsto which it is fused. The POI is at least 4 amino acids long, at least10 amino acids long or at least 20 amino acids long.

“Beta stem forming sequence” refers to the sequence of a passengerdomain of an autotransporter that forms a beta stem structure. The betastem forming sequence of a passenger can be identified using crystalstructure determination. As described above the beta stem formingsequence may alternatively be identified using the M4T homology modelingmethod (Rykunov et al 2009 J Struct Funct Genomics 10: 95-99) or similarprediction methods.

A “side domain” is a domain that is part of the passenger domain but isnot part of the beta stem. Typically, a side domain is located in thepassenger domain between two stretches of beta stem forming sequence. Aside domain starts at the first amino acid after the preceding betastrand and it ends one amino acid before the starting amino acid of thebeta strand following the side domain. The side domain can also belocated at the N-terminus of the passenger domain. Autotransporters mayhave several side domains.

“Similar protein”, “similar sequence” or a “like protein” refers to aprotein that has a high degree of homology to another protein when thetwo amino acid sequences are compared. Preferably, it is at least 80%,more preferably more than 90%, more preferably more than 95%, even morepreferably more than 97% homologous to the comparative sequence when thetwo sequences are optimally aligned. Sequence homology can be readilymeasured using public available software such as BLAST.

“Host cell” refers to a prokaryotic cell into which one or more vectorsor isolated and purified nucleic acid sequences of the invention havebeen introduced. It is understood that the term refers not only to theparticular subject cell but also to the progeny or potential progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutations or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

“Displayed”: A secreted protein is displayed on the surface of thesecreting host cell when it remains associated with the outer membraneof the host cell such that it at least partly protrudes outside thecell. The secreted protein may be attached to the cell membrane or acomponent that resides therein (such as the translocator domain from anautotransporter) in a covalent or non-covalent manner.

“Soluble secretion” and “secretion in a soluble manner” refers tosecretion of a protein where the protein is secreted into theextracellular space so that is not associated with the host cell asopposed to when the protein remains associated to the outer membrane ofthe host cell, or a protein that is integrated into the outer membraneof the host cell.

“Approximately” indicates a deviation of +/−10% of the stated value,where applicable.

DESCRIPTION

The inventors have found that an autotransporter protein can be used forimproved secretory protein expression if the beta stem forming sequenceof the passenger domain of the autotransporter is essentially intact.Whereas the actual beta stem-forming sequence is essential for optimalsecretion, the side domains of the passenger domain of autotransportersare suitable sites for the insertion of a POI. The side domains can bereplaced by a POI which will then be secreted. Alternatively, the POIcan be inserted so as to replace a part of the side domain or so as tobe fused to a side domain.

The inventors have also found that an autotransporter protein can beused for improved secretory protein expression of more than one, such asat least two, POI:s if the beta stem forming sequence of the passengerdomain of the autotransporter is essentially intact. By fusing, i.e.inserting, replacing or partly replacing, the POI:s to one or more sidedomains of the passenger, while keeping the beta stem structure intact,an efficient and relatively easy-to-use system for simultaneous displayor soluble secretion of two or more POI:s is achieved.

The side domains that can be replaced according to the invention arerelatively large, such as 20, 30, 40, 60, 80 or more amino acidresidues.

Thus, the passenger domain of an autotransporter can be considered asseveral sections of beta stem forming sequence linked together bynon-beta stem forming sequences. These non-beta stem forming sequencesare suitable sites for insertion of one or more POI:s. Thus, the POI canbe placed in between two parts of beta stem forming sequence. The POIcan also be fused to the N-terminus of the passenger domain.

Suitable methods for detecting beta stem forming sequence and sidedomains of passenger domains of autotransporters include biophysicalmethods such as as x-ray crystallography and bioinformatics softwaresuch as structure prediction tools.

X-ray crystallography is today a standard procedure that is highlyefficient and automatized and is known to a person skilled in the art.Examples of high resolution structures of passenger domains and suitablemethods for determination of structures of the passenger domain ofautotransporters are found in (Otto et al 2005 J Biol Chem280(17):17339-45; Emsley et al 1996 Nature 381: 90-92; Johnson et al2009 J Mol Biol 389(3): 559-74).

An example of a bioinformatics method that is suitable for determiningbeta stem structure is the M4T homology modeling method (Rykunov et al2009 J Struct Funct Genomics 10: 95-99), which is available for free onthe internet.

Where a three-dimensional model of the protein is used for theidentification of beta stem domains and side domains, it is suitablethat the model obtained has a resolution of better than 4 angstrom. Sidedomains will then be visible as domains that protrude from the betastem. By observation of the structure of the passenger domains ofautotransporters it can be seen that parts of the sequence are not partof the beta stem but form domains that protrude from the beta stem.

These methods can be used for determining which domains or amino acidsof the passenger domains that are suitable for insertion of a POI andwhich should be kept essentially intact.

The beta stem forming sequence is essentially intact according to theinvention. Thus, as little as possible of the beta stem forming sequenceshould be removed. Predicted domain border is of help to determine wherethe POI(s) should be inserted. If too much of the beta stem formingsequence is removed, secretion will be negatively affected. That thebeta stem forming sequence is essentially intact means that theefficiency of the secretory function of the protein is maintained at anoptimal level, as compared to when the beta stem is disrupted orcompletely removed. It also means that the stability of the passengerafter secretion is maintained. A person skilled in the art can useexperimental methods to determine if a particular constructs allowsefficient secretion.

Examples of methods suitable for determining the efficiency of secretionin vitro include: analysis of the fraction of POI present in the medium,labeling of surface proteins with biotin or other labels, cellfractionation, exposure of surface proteins to proteases (such asproteinase K) and studies using antibodies against the POI (such as dotblot studies, immunofluorescene microscopy and immuno-electronmicroscopy).

Examples of methods suitable for determining the stability of thepassenger after secretion include SDS-PAGE, western blotting and all ofthe above under paragraph 76.

Thus, by using structural information, a person skilled in the art canpredict where in the passenger domain the insertion of the POI can bemade in order to maintain optimal secretion. Actual secretion can beeasily determined with in vitro experiments.

According to the invention the POI is fused to the passenger domain.This means that the POI is fused to the peptide that forms the passengerdomain such that they form one continuous polypeptide. Because design ofthe fusion protein is carried out at the DNA level, care must be takenso that the reading frame of the POI is the same as the reading frame ofthe passenger domain.

Preferably the POI has a molecular weight of less than 200 kD. Morepreferably the molecular weight is less than 200 kD such as 100, 80, 60,40, 30, 20, 10 or 5 kD.

The fusion protein can comprise more than one POI. Thus, the fusionprotein can comprise two, three or more polypeptides of interest. Thefusion protein can be such that it has at least two POIs that eachreplaces, or partly replaces, or is fused to, a separate or independentside domain of the passenger domain. Alternatively, two or more POIs canbe fused to, or replace, or partly replace the same side domain.

The fusion protein is encoded by a nucleic acid and expressed in a hostcell. The nucleic acid can be constructed with the use of standardmolecular biology techniques involving restriction enzymes, DNA ligases,PCR, oligonucleotide synthesis, DNA purification and other methodswell-known to a person skilled in the art. Preferably, the startingpoint is a reading frame of an autotransporter protein into which a DNAfragment encoding the POI is inserted so that the reading frames match.Alternatively, the reading frame for the fusion protein can be designedin silico and synthesized using polynucleotide synthesis.

The reading frame encoding the fusion protein is preferably inserted inan expression vector for prokaryote expression carrying a promoter andother components well known to a person skilled in the art.

The fusion protein comprises an N-terminal signal peptide that directsthe protein for secretion. When the host cell is a Gram negativebacteria the signal peptide suitable is such that it directstranslocation of the protein across the inner membrane. The signalpeptide can be derived from an autotransporter protein, suitably thesame autotransporter from which the passenger domain is derived. Thesignal peptide can comprise approximately amino acids 1 to 52 of SEQ IDNO 1, or a similar sequence.

The fusion protein suitably comprises an autochaperone domain, suitablyfrom the passenger domain of the autotransporter protein used to fusethe POI. One example of an autochaperone domain comprises approximatelyamino acids 1002 to 1100 of SEQ ID NO 1.

The fusion protein can comprise a passenger domain from one type ofautotransporter and a translocator domain from another type ofautotransporter.

The autotransporter used in the invention can be an autotransporter witha serine protease domain, such as a serine protease.

The autotransporter can be a SPATE protein (Serine proteaseautotransporters of the Enterobacteriaceae). Thus, the translocatordomain and the passenger domain can be from a SPATE protein. In oneembodiment the SPATE protein is one of Hemoglobin-binding protease (Hbp)(SwissProt O88093) and temperature-sensitive hemagglutinin (Tsh)(SwissProt Q47692) from E. coli. The sequence of Tsh is homologous tothat of Hbp.

Other SPATE proteins include IgA protease of Neisseria gonorrhoeae andHaemophilus influenzae, EspC from E. coli, Pet from E. coli, EspP fromE. coli, Pic from E. coli, PicU from E. coli, Sat from E. coli, Vat fromE. coli, EspI from E. coli, EaaA from E. coli, EaaC from E. coli, EatAfrom E. coli, EpeA from E. coli, PssA from E. coli, AidA_B7A from E.coli, Boa from Salmonella bongori, SepA from Shigella flexneri, SigAfrom Shigella flexneri, Pic from Shigella flexneri.

The SPATE protein can comprise the polypeptide of SEQ ID NO 1, which isHbp, or SEQ ID NO 2, which is Hbp where the cleavage site between thetranslocator domain and the passenger domain has been disrupted (Hbpdelta-cleav) or a sequence that is similar to those sequences.Preferably the identity is more than 80%, even more preferably more than90%, even more preferably more than 95% and most preferably more than97% to those sequences.

The SPATE group of proteins has several advantages for use with thepresent invention. First of all some of their structures are known,facilitating the identification of their beta stem and side domains.This knowledge can also be used for prediction of side domains and betastem structures of related SPATEs for which the crystal structure is notknown. Another advantage is their cleavage structure that can be usedfor efficient soluble secretion, and that is conserved within the SPATEfamily.

Other autotransporters, for which the structure is known, can bepredicted or will be known, such that their beta stem and side domainstructure can be determined, may also be used with the presentinvention. The autotransporter should have a beta stem, a side domainand optionally a cleavage system that is efficient for solublesecretion. An example includes the autotransporter Hap_(s) from H.influenzae, which is not a member of the SPATE family. The structure ofthe passenger of Hap_(s) has recently been published (Meng et al 2011Aug. 12 The EMBO Journal, doi: 10.1038/emboj.2011.279. [Epub ahead ofprint]). The structure is very close to that of Hbp, having a beta-stemwith four side domains (SD1-4).

FIG. 11 A shows the crystal structure of the passenger domain of theautotransporter Hbp (Otto et al 2005 J Biol Chem 280(17): 17339-45).Domain 1 (d1), domain 2 (d2) and the autochaperone domain (ac) are inlight grey. The remainder of the passenger domain, including the betastem domain is colored black. Both domains d1 and d2 are suitable forinsertion of a POI. In addition, the domain d3 shown in FIG. 11 C anddomains d4 and d5 shown in FIG. 11D are suitable for replacement orinsertion of a POI.

Domain d1 comprises approximately the amino acids 53 to 308, d2comprises approximately the amino acids 533-608, d3 comprisesapproximately the amino acids 657-697, d4 comprises approximately theamino acids 735 to 766 and d5 comprises approximately amino acids 898 to922 of SEQ ID NO 1, which is the sequence of Hbp.

FIG. 11 E shows the domain composition of wild-type Hbp. In addition,fusion proteins used in the examples presented herein are shown. Inwild-type Hbp, the passenger domain comprising the beta stem (in black)and the side domains d1, d2, d3, d4 and d5 is shown. The translocatordomain is located at the C-terminal part of the protein and is indicatedas “β-domain”. “Ac” indicates an autochaperone domain. The signalpeptide is denoted by “ss”. Numbers indicate amino acid number from theN-terminus.

A passenger domain that comprises approximately amino acids 53-1100 ofSEQ ID NO 1, or a similar sequence, can be used.

A translocator domain that comprises approximately amino acids 1101-1377of SEQ ID NO1, or a similar sequence, can be used.

The POI can be a split protein. A split protein is a protein which inits native form comprises a single polypeptide or several polypeptidesthat are linked by disulphide bridges or other intermolecular bonds, andwhich for the present invention has been split in two or more parts.Each such part is fused to the passenger such that they form anon-native structure, for example at a distance apart. The two or moreparts may for instance be fused to different side domains or to the sameside domain but at a distance apart. Each such part is considered to beone POI, such that the split protein is considered to be two or morePOI:s. This could for example be advantageous when the native proteinhas a large or complex structure, for example comprising disulphidebridges, that inhibits efficient secretion. Splitting the protein maymake the secretion more efficient.

The POI can comprise at least one antigen, for example from aninfectious organism such as Mycobacterium tuberculosis. Examples of suchantigens from Mycobacterium tubercolosis include ESAT-6-like proteins(e.g. ESAT-6, TB10.4, TB10.3), an Ag85B-like protein (e.g. Ag85B), andRv 2660c. Two or more of such antigens may be fused to the samepassenger, for example to separate side domains.

ESAT-6 (early secretory antigenic target of 6 kDa) is a 10 kDa proteinthat is a potent T-cell antigen and an important virulence factor.

Rv2660c is a 7.6 kDa intracellular protein of unknown function.

TB10.3 and TB10.4 are both 96 amino acid proteins.

Ag85B is a secretory mycolyltransferase of 35 kDa, comprising threecysteins. It is also a potent T-cell antigen. This rather large andcysteine comprising protein is too complex, in its native form, foroptimal outer membrane translocation using the autotransporter system.

In one embodiment the antigen is split as defined above. For example,Ag85B, which is a large and rather complex protein, may be split into aN′-part (Ag85B(N′)) and a C′-part (Ag85B(C′)) for more efficientsecretion.

In one embodiment the POI comprises a polypeptide with a sequence thatis at least 80%, more preferably 90%, more preferably 95% mostpreferably 97% similar to SEQ ID NO 39, which is the sequence of ESAT-6.In one embodiment the POI comprises the polypeptide defined in SEQ ID NO39.

In one embodiment the POI comprises a polypeptide with a sequence thatis at least 80%, more preferably 90%, more preferably 95% mostpreferably 97% similar to SEQ ID NO 41, which is the sequence ofRv2660c. In one embodiment the POI comprises the polypeptide defined inSEQ ID NO 41.

In one embodiment the POI comprises a polypeptide with a sequence thatis at least 80%, more preferably 90%, more preferably 95% mostpreferably 97% similar to SEQ ID NO 42, which is the sequence of TB10.4.In one embodiment the POI comprises the polypeptide defined in SEQ ID NO42.

In one embodiment the POI comprises a polypeptide with a sequence thatis at least 80%, more preferably 90%, more preferably 95% mostpreferably 97% similar to SEQ ID NO 43, which is the sequence of TB10.3.In one embodiment the POI comprises the polypeptide defined in SEQ ID NO43.

In one embodiment the POI comprises a polypeptide with a sequence thatis at least 80%, more preferably 90%, more preferably 95% mostpreferably 97% similar to at least ¼ of SEQ ID NO 40, which is thesequence of Ag85B. In one embodiment the POI comprises the polypeptidedefined by amino acids 1-126 or 118-285 in SEQ ID NO 40.

The POI can be flanked by one or more linker regions. A linker regioncan be a flexible peptide of 1 to 20, or more, amino acids. The linkerregion can suitably be inserted at the C- and N-termini of the POI. Anadvantage of a linker is that it may allow the various domains of thefusion protein to move more independent of each other. A linker caneasily be designed by a person skilled in the art. Examples of suitablelinkers include SEQ ID NO 44 and 45.

The fusion protein can comprise the polypeptide defined in any of SEQ IDNO:s 13-19, SEQ ID NO:s 22-26 or SEQ ID NO 38 or a polypeptide which isat least 80%, more preferably 90%, more preferably 95% and mostpreferably 97% similar to any one of those sequences.

SEQ ID NO 13 is the sequence of Hbp were ESAT6 has replaced domain d1(Hbp(Δd1)-ESAT6, also named HbpSL-ESAT6). SEQ ID NO 14 is the sameprotein but where the cleavage site between the translocator domain andthe passenger domain has been disrupted (HbpD(Δd1)-ESAT6, also namedHbpDL-ESAT6). SEQ ID NO 15 is the sequence of Hbp where ESAT6 hasreplaced domain d2 (Hbp(Δd2)-ESAT6). SEQ ID NO 16 is the sequence of Hbpwhere ESAT6 has replaced domain d2 (HbpD(Δd2)-ESAT6, also namedHbpDD2-ESAT6) and where the cleavage site between the translocatordomain and the passenger domain has been disrupted. SEQ ID NO 17 is thesequence of Hbp where ESAT6 has replaced domain d3 (Hbp(Δd3)-ESAT6). SEQID NO 18 is the sequence of Hbp where ESAT6 has replaced domain d4(Hbp(Δd4)-ESAT6). SEQ ID NO 19 is the sequence of Hbp where ESAT6 hasreplaced domain d5 (Hbp(Δd5)-ESAT6).

SEQ ID NO 22 is the sequence of Hbp where Rv2660c has replaced domain d3(Hbp(Δd3)-Rv2660c). SEQ ID NO 23 is the sequence of Hbp where Rv2660chas replaced domain d4 (Hbp(Δd4)-Rv2660c). SEQ ID NO 24 is the sequenceof Hbp where Rv2660c has replaced domain d5 (Hbp(Δd5)-Rv2660c). SEQ IDNO 25 is the sequence of Hbp where TB10.4 has replaced domain d1(Hbp(Δd1)-TB10.4). SEQ ID NO 26 is the sequence of Hbp where TB10.3 hasreplaced domain d2 (Hbp(Δd2)-TB10.3).

SEQ ID NO 38 is the sequence of EspC where ESAT6 has replaced domain d1(EspC(Δd1)-ESAT6).

The fusion protein can comprise a polypeptide with more than one POI,such as the polypeptide defined in any of SEQ ID NO:s 28-35 or apolypeptide which is at least 80%, more preferably 90%, more preferably95% and most preferably 97% similar to any one of those sequences.

SEQ ID NO 28 is the sequence of Hbp where residues 1-126 of Ag85B hasreplaced domain d1 and residues 118-285 of Ag85B has replaced domain 2(Hbp-Ag85B_([N+C])). SEQ ID NO 29 is the sequence of Hbp where residues1-126 of Ag85B has replaced domain d1 and residues 118-285 of Ag85B hasreplaced domain 2, and where the cleavage site between the translocatordomain and the passenger domain has been disrupted (HbpD-Ag85B_([N+C])).SEQ ID NO 30 is the sequence of Hbp where residues 1-126 of Ag85B hasreplaced domain d2 and residues 118-285 of Ag85B has replaced domain 1(Hbp-Ag85B_([C+N])). SEQ ID NO 31 is the sequence of Hbp where residues1-126 of Ag85B has replaced domain d2 and residues 118-285 of Ag85B hasreplaced domain 1, and where the cleavage site between the translocatordomain and the passenger domain has been disrupted (HbpD-Ag85B_([C+N])).

SEQ ID NO 32 is the sequence of Hbp where residues 1-126 of Ag85B hasreplaced domain d2, residues 118-285 of Ag85B has replaced domain 1 andESAT6 has replaced domain d4 (Hbp-Ag85B_([C+N])-ESAT6). SEQ ID NO 33 isthe same protein but where the cleavage site between the translocatordomain and the passenger domain has been disrupted(HbpD-Ag85B_([C+N])-ESAT6). SEQ ID NO 34 is the sequence of Hbp whereresidues 1-126 of Ag85B has replaced domain d2, residues 118-285 ofAg85B has replaced domain 1, ESAT6 has replaced domain d4 and Rv2660chas replaced domain 5 (Hbp-Ag85B_([C+N])-ESAT6-Rv2660c). SEQ ID NO 35 isthe same protein but where the cleavage site between the translocatordomain and the passenger domain has been disrupted(HbpD-Ag85B_([C+N])-ESAT6-Rv2660c).

Preferably, the order of domains of the fusion protein is, from theN-terminus to the C-terminus: signal peptide, passenger domain,translocator domain.

In a second aspect of the invention it is provided a cell expressing afusion protein as defined herein. The cell is preferably a host cellthat can be cultured and manipulated by methods well known to a personskilled in the art and which is able to express heterologous proteins.Preferably the host cell is a Gram-negative bacterium such as E. coli,Salmonella spp., Vibrio spp., Shigella spp., Pseudomonads spp.,Burkholderia spp. or Bordetella spp. A wide variety of expressionsystems are available and known to a person skilled in the art. Theexpression may be of a stable or transient nature. The expression systemmay be inducible or non-inducible.

In one embodiment the fusion protein is at least partly solubly secretedby the host cell. This embodiment can be used when the invention is usedfor production of a recombinant protein, which is, for example, acommercial enzyme or a component of a pharmaceutical. The POI can thenbe conveniently harvested from the media, without breaking up the hostcells. Breaking up the host cells causes contamination with cellulardebris and cellular content. Secretion of the fusion protein can beachieved when the fusion protein comprises a protease cleavage sitebetween the translocator domain and the passenger domain. A proteaseactivity, which may reside in the fusion protein itself, cleaves thefusion protein when the translocator domain has integrated into theouter membrane so that the passenger domain is released into the medium.Alternatively, cleavage may take place via an intramolecularautocatalytic cleavage mechanism that is unrelated to protease activityas described for the SPATE EspP from E. coli (Dautin et al 2007 EMBO J.26(7): 1942-1952) and AIDA-I from E. coli (Charbonneau et al 2009 J BiolChem 284(25): 17340-17353).

For the sake of clearness, the POI may in some cases remain attached tothe cell membrane even though the polypeptide has been cleaved. Suchattachment will usually be of a non-covalent nature.

In one embodiment the POI remains covalently attached to thetranslocator domain. Where the sequence of the autotransporter harbors acleavage site, this can be achieved by mutating the cleavage sitebetween the translocator domain and the passenger domain, so that thecleavage event does not take place. Thus, the host cell displays atleast a part of the fusion protein comprising at least one POI on thecell surface.

In certain aspects the invention provides outer membrane vesicles(OMV:s) or bacterial ghosts displaying a fusion protein according to theinvention on their surface.

Under certain conditions Gram negative bacteria may be induced to startshedding vesicles from their outer membrane. Such outer membranevesicles (OMV:s) have for example been shown to be useful as vaccineplatforms. When carrying antigens, as derived from their mother cells,these vesicles are capable of enhancing the immunogenicity of suchantigen. OMV:s may easily be derived from gram negative bacteriadisplaying the fusion protein of the invention on their surface. Methodsfor outer membrane vesicle production and isolation are known in the art(Chen et al 2010 PNAS 107:3099-3104; Bernadac et al 1998 J Bacteriol180: 4872-4878; Kesty and Kuehn 2004 J Biol Chem 279: 2069-2076);Kolling and Matthews 1999 App Env Microbiol 65: 1843-1848; Kitagawa etal 2010 J Bacteriol 192: 5645-5656).

Similarly, bacterial ghosts are a nonliving vaccine platform. Bacterialghosts are bacterial cell envelopes that have been emptied of theircytoplasm by means of lysis, for example using the lethal lysis gene Efrom bacteriophage PhiX174 (Langemann et al 2010 Bioeng Bugs 1:326-336;Young 1992 Microbiol rev 56: 430-481; Mayr et al 2005 Adv Drug Deliv rev57: 1381-1391). They retain all morphological, structural and antigenicfeatures of the mother cell and comprise proteins that are expressed andanchored to the cell envelope before lysis. Delivery of for exampleantigenic proteins can be facilitated by the secretion system and thefusion proteins of the invention.

One aspect of the invention is a vaccine comprising a fusion protein, acell, an outer membrane vesicle or a bacterial ghost according to theinvention. The vaccine can comprise a host cell that displays a fusionprotein comprising at least one POI at the cell surface. Preferably thePOI is then an antigen as described above. The host cell can be anattenuated Salmonella strain, such as the strains described in Curtiss R3^(rd) et al 2010 Crit Rev Immunol 30(3): 255-70. The vaccine cancomprise living host Salmonella cells.

One aspect of the invention is a nucleic acid which encodes a fusionprotein according to the invention as has been described above. Onefurther aspect of the invention is a vector carrying a nucleic acidaccording to the invention.

The nucleic acid or vector may be arranged for expression of more thanone POI fused to the same passenger domain. For example, the sequencewhich encodes the passenger domain can comprise at least two stretchesof cloning site sequence that allow in-frame cloning of at least two POIencoding sequences. This facilitates easy cloning and expression of anydesired POI:s. Alternatively the nucleic acid may comprise more than onesequence encoding POI:s, fused to the passenger domain.

One aspect of the invention comprises a method for secretory proteinexpression of a POI comprising the step of expressing a fusion proteinaccording to the invention in a host cell. Expression vectors are wellknown to a person skilled in the art. Suitably, the vector has apromoter suitable for the host cell which is operatively linked to thenucleic acid that encodes the fusion protein according to the invention.

The method can comprise the step of identifying suitable side domains onan autotransporter protein. This can be carried out with the biophysicalmethods or the bioinformatics methods described above.

One aspect of the method according to the invention comprises the stepof replacing a side domain (or a part thereof) of a passenger domain ofan autotransporter with a POI so that the beta-stem forming sequence ofthe passenger domain of the autotransporter is essentially intact.Alternatively, the method can comprise the step of inserting the POIinto the passenger domain so that the beta stem forming sequence isessentially intact.

The method comprises the step of culturing the host cell underconditions wherein the nucleic acid encoding the fusion protein istranslated to a multitude of fusion protein molecules and the fusionprotein molecule enters the secretory pathway.

In one embodiment, the method comprises the additional step ofinhibiting a periplasmic enzyme with protease activity in the host cell,such as DegP. The protease activity of DegP can be inhibited bydeleting, interrupting or inactivating the DegP-encoding gene on thechromosome of the host cell. Inactivation can be carried out by theintroduction of a mutation in the catalytic site of DegP. The inhibitionof a protease has the advantage that yield can be improved.

In one embodiment the method comprises the additional step of downregulation of at least one enzyme, such as DsbA or DsbB, that catalysesthe formation of disulphide bonds in proteins in the periplasmic spaceof the host cell. This has the advantage that yield can be improved,especially for proteins that are prone to form disulphide bridges, suchas proteins of eukaryotic origin.

In one embodiment of the method the POI is soluble secreted. In oneembodiment of the method the POI remains covalently attached to the cellsurface.

In one embodiment the method comprises the further step of inducingshedding of vesicles from the outer membrane of the host cell, toproduce outer membrane vesicles displaying the fusion protein of theinvention on their surface.

In another embodiment the method comprises the additional step of lysingthe gram negative bacterium, for example using the lethal lysis gene Efrom bacteriophage PhiX174, thus forming bacterial ghosts displaying thefusion protein on their surface.

One final aspect of the invention comprises a fusion protein obtainableaccording to the method of the invention.

EXAMPLES Methods

Strains and Media

E. coli strain MC1061 (araD139 Δ(araA-leu)7697 ΔlacX74 galK16galE15(GalS) λ⁻ e14⁻ mcrA0 relA1 rpsL150(strR) spoT1 mcrB1 hsdR2) hasbeen described previously (Casadaban and Cohen 1980 J Mol Biol 138:179-207). Strain TOP10F′ was obtained from Invitrogen.

Cells were routinely grown at 37° C. in LB medium supplemented with 0.2%glucose. Overnight cultures were grown in the presence of 0.4% glucose.Cells were grown in the presence of chloroamphenicol (30 μg/ml) andstreptomycin (25 μg/ml) or Tetracycline (6.25 μg/ml), where appropriate.

Construction of Plasmids

Plasmid pEH3-Hbp (FIG. 1) carries the full-length hbp gene, theexpression of which is under control of an inducible LacUV5 promoter.The construction of this plasmid has been described in (Jong et al 2007Mol Microbiol 63(5): 1524-1536).

In FIGS. 1-10 the translocator domain is referred to as “β-domain”.

TABLE 1 Primers used in this study SEQ ID Name NO Sequence (5′ à 3′)Hbp944-962 fw  86 gaacatcggaaggtggtgc Hbp1123-1104 rv  87gagaaaccgaatccttaagg Hbp2154-2137 rv  88 ggatggttgtgttcagtgtgpEH_XbaI_Hbp fw  89taactttctagattacaaaacttaggagggtttttaccatgaacagaatttattctcttcgEcoRI_Hbp rv  90 cagtgaattctcagaatgaataacgaatattag Hbp(Δdom1/Cas) fw  91gggagctcctgcggatccggcagcggtaatgatgccccggtcacgttc Hbp(Δdom1/Cas) rv  92cggatccgcaggagctccccgcaagacttcctgcagag Hbp(Δdom2/Cas) fw  93ctgggagctccgcaggatccggcagcggtaatactgcagggtatctgtttc Hbp(Δdom2/Cas) rv 94 ctgccggatcctgcggagctcccagaaccggcatagtccagcgtgatagHbp(Δβ-stem/Cas) fw  95 gggagctcctgcggatccggcagcggtgcagacaaactggtgataaacHbp 2838-2820 rv  96 gttcatcgaccactgggtg Hbp(Δdom3/Cas) fw  97gggagcgggagctccgcaggatccggcagcggtaaccgcagttttacctttgac Hbp(Δdom3/Cas) rv 98 accgctgccggatcctgcggagctcccgctcccctgcagcgtcagacg Hbp 1859-1879 fw 99 gcaatctgaatgtggacaatc Hbp(Δdom4/Cas) fw 100Gggagcgggagctccgcaggatccggcagcggtagtgtcttcaacggcaccg Hbp(Δdom4/Cas) rv101 accgctgccggatcctgcggagctcccgctcccgtcgcccagcgtgacgctgHbp(Δdom5/Cas) fw 102GggagcgggagctccgcaggatccggcagcgGGTACCgcaatatctggagc Hbp(Δdom5/Cas) rv103 gctgccggatcctgcggagctcccgctccctccgagggtgacagtc Hbp 3003-3021 rv 104gtcatgacctgttgccgac Hbp(d4ins/Cas) fw 105ctgggagctccgcaggatccggcagcggtaaaagtgtcttcaacggcacc Hbp(d4ins/Cas) rv 106ctgccggatcctgcggagctcccagaacctgcaacagatgtgccttcttc Hbp((βins/Cas) fw 107GggagcgggagctccgcaggatccggcagcggtaccgtcaacctggataatcagtHbp((βins/Cas) rv 108accgctgccggatcctgcggagctcccgctcccgccgttgaagacacttttatctg Cas/Rv2660c fw109 cggggagctccgtgatagcgggcgtcgacc Cas/Rv2660c rv 110tgccggatccgtgaaactggttcaatcccag Cas/TB10.4 fw 111cggggagctccatgtcgcaaatcatgtacaac Cas/TB10.4 rv 112tgccggatccgccgccccatttggcgg Cas/Ag85B fw 113 cggggagctccttctcccggccggggcCas/Ag85B rv 114 tgccggatccgccggcgcctaacgaac Cas/Ag85B(T118) fw 115cggggagctccaccggcagcgctgcaatcg Cas/Ag85B(S126) rv 116tgccggatcccgacaagccgattgcagcg pEH_XbaI_EspC_fw 117taactttctagattacaaaacttaggagggtttttaccatgaataaaatatacgcattaaaataEcoRI_EspC rv 118 Gtcagaattctcagaaagaataacggaagttag EspC(Δdom1/Cas) fw119 gggagctccgcaggatccggcagcggtttaaaaaacaaatttactcaaaaagtcEspC(Δdom1/Cas) rv 120 cggatcctgcggagctcccagcctgagatgcgcttaaaaaagEspC (BglII) rv 121 Ccagagccaatgtttacgtc p 15a fw 122gtacgaattcgtgcgtaacggcaaaagcac p15a rv 123gtacgtcgacacatgagcagatcctctacg

Plasmid pEH3-Hbp[Δβ-cleav] (FIG. 2) is a pEH3-Hbp (Jong et al, 2007)derivative that carries an hbp mutant that encodes a version of Hbp inwhich the natural cleavage site between the passenger domain and thetranslocator domain has been disrupted upon substitution of amino acidresidues Asn¹¹⁰⁰ and Asn¹¹⁰¹ by a Gly and Ser residue, respectively. Theconstruction of pEH3-Hbp[Δβ-cleav] has been described in (Jong et al2007 Mol Microbiol 63(5): 1524-1536).

Plasmid pHbpD(Δd1), which is the same as pHbpDL, (FIG. 3) is apEH3-Hbp[Δβ-cleav] (Jong et al 2007 Mol Microbiol 63(5): 1524-1536)derivative that carries an hbp mutant that encodes a truncated versionof Hbp[Δβ-cleav] (Jong et al 2007 Mol Microbiol 63(5): 1524-1536) inwhich amino acid residues 54-307 of the full-length Hbp amino acidsequence have been replaced by the amino acid sequenceSer-Ser-Cys-Gly-Ser-Gly-Ser-Gly (SEQ ID NO 45). The DNA sequence thatencodes the latter amino acid sequence contains SacI and BamHIrestriction sites that allow easy in-frame cloning of DNA sequences thatencode heterologous amino acid sequences into the HbpD(Δd1) codingsequence. To create pHbpD(Δd1), first, a variant of pEH3-Hbp[Δβ-cleav](pEH3-Hbp[Δβ-cleav/ΔBamHI]) was created lacking BamHI restriction sitesinside and outside of the Hbp[Δβ-cleav] coding region, respectively.Subsequently, a three-step ‘overlapping extension PCR’ procedure wascarried out. In the first step a DNA fragment was amplified by PCR usingpEH3-Hbp (Jong et al 2007 Mol Microbiol 63(5): 1524-1536) as a templateand the primers pEH_XbaI_Hbp fw and Hbp(Δdom1/Cas) rv. In the secondstep a DNA fragment was amplified by PCR using pEH3-Hbp (Jong et al 2007Mol Microbiol 63(5): 1524-1536) as a template and the primersHbp(Δdom1/Cas) fw and Hbp1123-1104 rv. In the third step a DNA fragmentwas amplified using a mixture of the PCR products from step 1 and 2 astemplate and the primers pEH_XbaI_Hbp fw and Hbp1123-1104 rv. The PCRproduct from step three was cloned into pEH3-Hbp[Δβ-cleav/ΔBamHI] usingthe XbaI and NdeI restriction sites, yielding plasmid pHbpD(Δd1).

For primers used in this study see Table 1.

Plasmid pHbpD(Δd2), which is the same as pHbpDD2, (FIG. 5) was createdaccording to the same general procedure as pHbpD(Δd1), but with thefollowing modifications: Amino acid residues 534-607 of the full-lengthHbp amino acid sequence was replaced by the amino acid sequenceGly-Ser-Gly-Ser-Ser-Ala-Gly-Ser-Gly-Ser-Gly (SEQ ID NO 44). The DNAsequence that encodes the amino acid sequence also contains SacI andBamHI restriction sites for easy in-frame cloning of DNA sequences thatencode heterologous amino acid sequences. For the first PCRamplification step primers Hbp944-962 fw and Hbp(Δdom2/Cas) rv wereused. For the second PCR amplification step primers Hbp(Δdom2/Cas) fwand Hbp 2154-2137 rv were used. And for the third step primersHbp944-962 fw and Hbp2154-2137 rv were used. The PCR product from stepthree was cloned into pEH3-Hbp[Δβ-cleav/ΔBamHI] using NdeI and NsiIrestriction sites.

Plasmid pHbp(Δd1), which is the same as pHbpSL, (FIG. 7) is a pEH3-Hbp(Jong et al 2007 Mol Microbiol 63(5): 1524-1536) derivative that carriesan hbp mutant that encodes a truncated version of Hbp [pHbp(Δd1)] inwhich amino acid residues 54-307 of the full-length Hbp amino acidsequence have been replaced by the amino acid sequenceSer-Ser-Cys-Gly-Ser-Gly-Ser-Gly (SEQ ID NO 45). The DNA sequence thatencodes the latter amino acid sequence contains SacI and BamHIrestriction sites that allow easy in-frame cloning of DNA sequences thatencode heterologous amino acid sequences into the Hbp(Δd1) codingsequence. To construct pHbp(Δd1), first a variant of pEH3-Hbp(pEH3-Hbp/4BamHI) was created lacking a BamHI site downstream of the hbpORF. Subsequently, a three-step ‘overlapping extension PCR’ procedurewas carried out. In the first step a DNA fragment was amplified by PCRusing pEH3-Hbp (Jong et al 2007 Mol Microbiol 63(5): 1524-1536) as atemplate and the primers pEH_XbaI_Hbp fw and Hbp(Δdom1/Cas) rv. In thesecond step a DNA fragment was amplified by PCR using pEH3-Hbp (Jong etal 2007 Mol Microbiol 63(5): 1524-1536) as a template and the primersHbp(Δdom1/Cas) fw and Hbp1123-1104 rv. In the third step a DNA fragmentwas amplified using a mixture of the PCR products from step 1 and 2 astemplate and the primers pEH_XbaI_Hbp fw and Hbp1123-1104 rv. The PCRproduct from step three was cloned into pEH3-Hbp[ΔBamHI], a derivativeof pEH3-Hbp lacking a BamHI restiction site downstream of the hbp gene,using the XbaI and NdeI restriction sites, yielding plasmid pHbp(Δd1).

Plasmids pHbpSS (FIG. 9), pHbp(Δd2), pHbp(Δd3), pHbp(Δd4), pHbp(Δd5),pHbp(d4ins) and pHbp(βins) were created according to the same generalprocedure as pHbpD(Δd1), but with the following modifications:

For pHbpSS: Amino acid residues 54-993 of the full-length Hbp amino acidsequence were replaced by the amino acid sequenceSer-Ser-Cys-Gly-Ser-Gly-Ser-Gly (SEQ ID NO 45). For the first PCRamplification step primers pEH_XbaI_Hbp fw and Hbp(Δdom1/Cas) rv wereused. For the second PCR amplification step primers Hbp(Δβ-stem/Cas) fwand EcoRI_Hbp rv were used. And for the third step primers pEH_XbaI_Hbpfw and EcoRI_Hbp rv were used. The PCR product from step three wascloned into pEH3-Hbp[ΔBamHI] using the XbaI and NdeI restriction sites,yielding plasmid pHbpSS.

For pHbp(Δd2): Amino acid residues 534-607 of the full-length Hbp aminoacid sequence were replaced by the amino acid sequenceGly-Ser-Gly-Ser-Ser-Ala-Gly-Ser-Gly-Ser-Gly (SEQ ID NO 44), thecorresponding DNA sequence of which contains SacI and BamHI restrictionsites for easy in-frame cloning of DNA sequences. For the first PCRamplification step primers Hbp944-962 fw and Hbp(Δdom2/Cas) rv wereused. For the second PCR amplification step primers Hbp(Δdom2/Cas) fwand Hbp 2154-2137 rv were used. And for the third step primersHbp944-962 fw and Hbp2154-2137 rv were used. The PCR product from stepthree was cloned into pEH3-Hbp[ΔBamHI] using the NdeI and NsiIrestriction sites, yielding plasmid pHbp(Δd2).

For pHbp(Δd3): Amino acid residues 659-696 of the full-length Hbp aminoacid sequence were replaced by the amino acid sequenceGly-Ser-Gly-Ser-Ser-Ala-Gly-Ser-Gly-Ser-Gly (SEQ ID NO 44). For thefirst PCR amplification step primers Hbp944-962 fw and Hbp(Δdom3/Cas) rvwere used. For the second PCR amplification step primers Hbp(Δdom3/Cas)fw and Hbp 2838-2820 rv were used. And for the third step primersHbp944-962 fw and Hbp 2838-2820 rv were used. The PCR product from stepthree was cloned into pEH3-Hbp[ΔBamHI] using the NdeI and KpnIrestriction sites, yielding plasmid pHbp(Δd3).

For pHbp(Δd4): Amino acid residues 736-765 of the full-length Hbp aminoacid sequence were replaced by the amino acid sequenceGly-Ser-Gly-Ser-Ser-Ala-Gly-Ser-Gly-Ser-Gly (SEQ ID NO 44). For thefirst PCR amplification step primers Hbp1859-1879 fw and Hbp(Δdom4/Cas)rv were used. For the second PCR amplification step primersHbp(Δdom4/Cas) fw and Hbp 2838-2820 rv were used. And for the third stepprimers Hbp1859-1879 fw and Hbp2838-2820 rv were used. The PCR productfrom step three was cloned into pEH3-Hbp[ΔBamHI] using the NsiI and KpnIrestriction sites, yielding plasmid pHbp(Δd4).For pHbp(Δd5): Amino acid residues 899-920 of the full-length Hbp aminoacid sequence were replaced by the amino acid sequenceGly-Ser-Gly-Ser-Ser-Ala-Gly-Ser-Gly-Ser-Gly (SEQ ID NO 44). For thefirst PCR amplification step primers Hbp1859-1879 fw and Hbp(Δdom5/Cas)rv were used. For the second PCR amplification step primersHbp(Δdom5/Cas) fw and Hbp3003-3021 rv were used. And for the third stepprimers Hbp1859-1879 fw and Hbp3003-3021 rv were used. The PCR productfrom step three was cloned into pEH3-Hbp[ΔBamHI] using the NsiI and KpnIrestriction sites, yielding plasmid pHbp(Δd5).

For pHbp(d4ins): Amino acid residues 760-764 of the full-length Hbpamino acid sequence were replaced by the amino acid sequenceGly-Ser-Gly-Ser-Ser-Ala-Gly-Ser-Gly-Ser-Gly (SEQ ID NO 44). For thefirst PCR amplification step primers Hbp1859-1879 fw and Hbp(d4ins/Cas)rv were used. For the second PCR amplification step primersHbp(d4ins/Cas) fw and Hbp 2838-2820 rv were used. And for the third stepprimers Hbp1859-1879 fw and Hbp2838-2820 rv were used. The PCR productfrom step three was cloned into pEH3-Hbp[ΔBamHI] using the NsiI and KpnIrestriction sites, yielding plasmid pHbp(d4ins).

For pHbp(βins): Amino acid sequenceGly-Ser-Gly-Ser-Ser-Ala-Gly-Ser-Gly-Ser-Gly (SEQ ID NO 44) was insertedbetween residues 771 and 772 of the full-length Hbp amino acid sequence.For the first PCR amplification step primers Hbp1859-1879 fw andHbp(βins/Cas) rv were used. For the second PCR amplification stepprimers Hbp(βins/Cas) fw and Hbp 2838-2820 rv were used. And for thethird step primers Hbp1859-1879 fw and Hbp2838-2820 rv were used. ThePCR product from step three was cloned into pEH3-Hbp[ΔBamHI] using theNsiI and KpnI restriction sites, yielding plasmid pHbp(βins).

ESAT6 derivatives of the plasmids above were derived by a heterologousinsertion corresponding to the Mycobacterium tuberculosis ESAT6 proteininto the respective plasmids. To construct the ESAT6 derivatives asynthetic ESAT6-encoding DNA sequence was obtained from BaseClear B.V.(Leiden, The Netherlands), the codon-usage of which was optimized forexpression in E. coli. The synthetic DNA fragment possessed SacI andBamHI sites at the 5′ and 3′ side of the ESAT6 coding sequence,respectively. This allowed cloning into the SacI and BamHI sites ofpHbpD(Δd1), pHbpD(Δd2), pHbp(Δd1), pHbpSS, pHbp(Δd2), pHbp(Δd3),pHbp(Δd4), pHbp(Δd5), pHbp(d4ins) and pHbp(βins), yieldingpHbpD(Δd1)-ESAT6, which is the same as pHbpDL-ESAT6 (FIG. 4),pHbpD(Δd2)-ESAT6, which is the same as pHbpDD2-ESAT6 (FIG. 6),pHbp(Δd1)-ESAT6, which is the same as pHbpSL-ESAT6 (FIG. 8),pHbpSS-ESAT6 (FIG. 10), pHbp(Δd2)-ESAT6, pHbp(Δd3)-ESAT6,pHbp(Δd4)-ESAT6, pHbp(Δd5)-ESAT6, pHbp(d4ins)-ESAT6 andpHbp(pins)-ESAT6, respectively.

Rv2660c derivatives of plasmids above were derived by a heterologousinsertion corresponding to the Mycobacterium tuberculosis Rv2660cprotein into the respective plasmids. To construct the Rv2660cderivatives the gene encoding Rv2660c with flanking SacI/BamHI sites wasamplified by PCR using M. tuberculosis H37Rv genomic DNA as a template.The primers used were Cas/Rv2660c fw and Cas/Rv2660c rv. The PCR productwas cloned into pHbp(Δd3), pHbp(Δd4) and pHbp(Δd5) using the SacI/BamHIsites, creating pHbp(Δd3)-Rv2660c, pHbp(Δd4)-Rv2660c andpHbp(Δd5)-Rv2660c, respectively.

TB10.3 and TB10.4 derivatives of plasmids above were derived by aheterologous insertion corresponding to the Mycobacterium tuberculosisproteins TB10.3 or TB10.4 into the respective plasmids. To constructTB10.3 and TB10.4 derivatives, the gene encoding TB10.3 or TB10.4 withflanking SacI/BamHI sites were amplified by PCR using M. tuberculosisH37Rv genomic DNA as a template. The primers used for TB10.3 wereCas/TB10.3 fw and Cas/TB10.3 rv. The PCR product was cloned intopHbp(Δd2) using the SacI/BamHI sites, creating pHbp(Δd2)-TB10.3. Theprimers used for TB10.4 were Cas/TB10.4 fw and Cas/TB10.4 rv. The PCRproduct was cloned into pHbp(Δd1) using the SacI/BamHI sites, creatingpHbp(Δd1)-TB10.4.

Plasmid pHbp(Δd1)-hEGF(0ss) is a pHbp(Δd1) derivative expressingHbp(Δd1) containing a heterologous insertion corresponding to acysteineless version of the Homo sapiens hEGF protein. To constructpHbp(Δd1)-hEGF(0ss) a synthetic hEGF(0ss) encoding DNA sequence wasobtained possessing SacI and BamHI sites at the 5′ and 3′ side of thehEGF(0ss) coding sequence, respectively, to allow cloning into the SacIand BamHI sites of pHbp(Δd1), yielding pHbp(Δd1)-hEGF(0ss).

Plasmid pHbp-Ag8513_((N+C)) is a pEH3-Hbp/ΔBamHI derivative expressing amutant of Hbp in which an amino acid sequence corresponding to residues1-126 of the mature region of the protein Ag85B from Mycobacteriumtuberculosis (Ag85B_((N′))) was inserted into a flexible linker that waslocated as described for pHbp(Δd1). In addition, an amino acid sequencecorresponding to residues 118-285 of the mature region of the proteinAg85B (Ag8513_((C′))) was inserted into a flexible linker that waslocated as described for pHbp(Δd2). To construct pHbp-Ag8513_((N+C)),fragments of fbpA encoding Ag85B_((N)) and Ag85B_((C)) were generatedwith flanking SacI/BamH sites using M. tuberculosis H37Rv genomic DNA asa template. For Ag85B_((N)), the primers used were Cas/Ag85B fw andCas/Ag85B(S126) rv. The resulting PCR fragment was cloned into pHbp(Δd1)using the SacI/BamHI restriction sites, creating pHbp(Δd1)-Ag85B_((N)).For Ag85B_((C)) the primers used were Cas/Ag85B(T118) fw and Cas/Ag85Brv. The resulting PCR fragment was inserted into pHbp(Δd2) using theSacI/BamHI restriction sites, creating pHbp(Δd2)-Ag85B_((C)).Subsequently, the XbaI/NdeI fragment of pHbp(Δd2)-Ag85B_((C)) wassubstituted by the XbaI/NdeI fragment of pHbp(Δd1)-Ag85B_((N)), yieldingpHbp-Ag85B_((N+C)).

Plasmids pHbp-Ag85B_((C+N)), pHbpD-Ag85B_((N+C)) and pHbpD-Ag85B_((C+N))were created according to the same general procedure aspHbp-Ag85B_((N+C)), but with the following modifications:

For pHbp-Ag85B_((C+N)) the N-terminal part (residues 1-126) of themature region of the protein Ag85B from Mycobacterium tuberculosis(Ag85B_((N′))) was inserted into a flexible linker that was located asdescribed for pHbp(Δd2) and the C-terminal part (residues 118-285) wasinserted into a flexible linker that was located as described forpHbp(Δd1). After PCR, using the same primers as above, the Ag85B_((N))PCR product was cloned into pHbp(Δd2) using the SacI/BamHI restrictionsites, and the Ag85B_((C)) PCR product was cloned into pHbp(Δd1) usingthe SacI/BamHI restriction sites, creating pHbp(Δd2)-Ag85B_((N)) andpHbp(Δd1)-Ag85B_((C)) respectively. Subsequently, the XbaI/NdeI fragmentof pHbp(Δd2)-Ag85B_((N)) was substituted by the XbaI/NdeI fragment ofpHbp(Δd1)-Ag85B_((C)), yielding pHbp-85B_((C+N)).

For pHbpD-Ag85B_((N+C)) and pHbpD-Ag85B_((C+N)) the same procedures asfor pHbpD-Ag85B_((N+C)) and pHbpD-Ag85B_((C+N)) respectively were used,except that plasmids pHbpD(Δd1) and pHbpD(Δd2) were used instead ofplasmids pHbp(Δd1) and pHbp(Δd2).

Plasmid pHbp-Ag85B_([C+N])-ESAT6 is derivative of pHbp-Ag85B_([C+N])encoding a version of Hbp-Ag85B_([C+N]) in which an amino acid sequencecorresponding to ESAT6 was inserted into a flexible linker that waslocated as described for pHbp(d4ins). To constructpHbp-Ag85B_([C+N])-ESAT6, the NsiI/KpnI fragment of pHbp-Ag85B_([C+N])was substituted by that of pHbp(d4 in)-ESAT6, creatingpHbp-Ag85B_([C+N])-ESAT6. Plasmid pHbpD-Ag85B_([C+N])-ESAT6 was createdcorrespondingly, except that the NsiI/KpnI fragment ofpHbpD-Ag85B_([C+N]) was substituted by that of pHbp(d4 in)-ESAT6.

Plasmid pHbp-Ag85B_([C+N])-ESAT6-Rv2660c is derivative ofpHbp-Ag85B_([C+N])-ESAT6 encoding a version of Hbp-Ag85B_([C+N])-ESAT6in which an amino acid sequence corresponding to Rv2660c was insertedinto a flexible linker that was located as described for pHbp(Δd5). Toconstruct pHbp-Ag85B_([C+N])-ESAT6-Rv2660c, the BstZ17i/KpnI fragment ofpHbp-Ag85B_((C+N))-ESAT6 was substituted by that of pHbp(Δd5)-Rv2660c,yielding pHbp-Ag85B_((C+N))-ESAT6-Rv2660c. PlasmidpHbpD-Ag85B_((C+N))-ESAT6-Rv2660c was created correspondingly, exceptthat the BstZ17i/KpnI fragment of pHbpD-Ag85B_((C+N))-ESAT6 wassubstituted by that of pHbp(Δd5)-Rv2660c.

Plasmid pEH3-EspC carries the full-length espC gene, the expression ofwhich is under control of an inducible LacUV5 promoter. To constructpEH3-EspC, the espC gene was amplified by PCR using pJLM174 (Dutta et al2002 Infect. Immun. 70, 7105-7113) as a template and the primerspEH_XbaI_EspC fw and EcoRI_EspC rv. The resulting PCR product was clonedinto the XbaI/EcoRI sites of pEH3-Hbp. This step effectively exchangedthe espC ORF for that of hbp, resulting in pEH3-EspC.

Plasmid pEH3-EspC(Δd1) is a pEH3-EspC derivative that carries an espCmutant that encodes a truncated version of EspC in which amino acidresidues 54-300 of the full-length EspC amino acid sequence have beenreplaced by the amino acid sequence Gly-Ser-Ser-Ala-Gly-Ser-Gly-Ser-Gly(SEQ ID NO 46). The DNA sequence that encodes the latter amino acidsequence contains SacI and BamHI restriction sites that allow easyin-frame cloning of DNA sequences that encode heterologous amino acidsequences into the EspC(Δd1) coding sequence. To create pEH3-EspC(Δd1),a three-step ‘overlapping extension PCR’ procedure was carried out. Inthe first step a DNA fragment was amplified by PCR using pEH3-EspC as atemplate and the primers pEH_XbaI_EspC fw and EspC(Δdom1/Cas) rv. In thesecond step a DNA fragment was amplified by PCR using pEH3-EspC as atemplate and the primers EspC(Δdom1/Cas) fw and EspC(BgIII) rv. In thethird step a DNA fragment was amplified using a mixture of the PCRproducts from step 1 and 2 as template and the primers pEH_XbaI_EspC fwand EspC(BgIII) rv. The PCR product from step three was cloned intopEH3-EspC using the XbaI and BgIII restriction sites, yielding plasmidpEH3-EspC(Δd1).

Plasmid pEH3-EspC(Δd1)-ESAT6 is a pEH3-EspC(Δd1) derivative expressingEspC(Δd1) containing a heterologous insertion corresponding to themycobacterium tuberculosis ESAT6 protein. To constructpEH3-EspC(Δd1)-ESAT6 a synthetic ESAT6-encoding DNA sequence possessingSacI and BamHI sites at the 5′ and 3′ side was obtained as describedabove. This allowed cloning of the synthetic ESAT6-encoding DNA sequenceinto the SacI and BamHI sites of pEH3-EspC(Δd1), yieldingpEH3-EspC(Δd1)-ESAT6.

Plasmid pEH3_((p15a))-HbpD-Ag85B_([C+N])-ESAT6 (FIG. 34) is identical toplasmid pHbpD-Ag85B_([C+N])-ESAT6 except that a DNA fragment carryingthe pMB1 origin of replication has been replaced by a fragment carryinga p15a origin of replication. To createpEH3_((p15a))-HbpD-Ag85B_([C+N])-ESAT6, plasmid pEH3_((p15a))-Hbp wascreated first. This plasmid was generated using pBAD33 (Guzman et al1995 Journal of Bacteriology 177:4121-4130) as a template and theprimers p15a fw and p15a rv (see Table 1). The resulting PCR fragment,carrying the p15a origin of replication, was cloned into pEH3-Hbp usingthe SalI/EcoRI restriction sites, yielding pEH3_((p15a))-Hbp.Subsequently, the XbaI/EcoRI fragment of pEH3_((p15a))-Hbp wassubstituted by that of pHbpD-Ag85B_([C+N])-ESAT6, creatingpEH3_((p15a))-HbpD-Ag85B_([C+N])-ESAT6.

Description of Constructs Used in the Examples

For more detailed descriptions and how the constructs were made, seeabove under “Construction of plasmids”.

FIG. 11 A shows the crystal structure of the passenger domain of theautotransporter Hbp (Otto et al 2005 J Biol Chem 280(17): 17339-45).Domain 1 (d1), domain 2 (d2) and the autochaperone domain (ac) are inlight grey. The remainder of the passenger, including the beta stemdomain is colored black.

FIG. 11 B shows the crystal structure of the passenger domain of theautotransporter Hbp (Otto et al 2005 J Biol Chem 280(17): 17339-45),rotated around the y-axis (50° counter clockwise) compared to thesituation depicted in FIG. 11 A. The image was created using MacPyMol.

FIG. 11 C shows the crystal structure of the passenger domain of theautotransporter Hbp as in FIG. 11 B, but the residues that comprisedomain 1 are hidden. Domain 3 (d3) is depicted in light grey. Theremainder of the passenger is coloured black. The image was createdusing MacPyMol.

FIG. 11 D shows the crystal structure of the passenger domain of theautotransporter Hbp as in FIG. 11 C. Domain 4 (d4) and a side domainthat corresponds to residues 898-922 (d5) of Hbp are depicted in lightgrey. The remainder of the passenger is coloured black. The image wascreated using MacPyMol.

FIG. 11 E shows schematic representations of Hbp-derivative constructsused in the examples. For the examples disclosed herein theHbp-derivative constructs shown in FIG. 11 E were cloned into plasmidspEH3-Hbp[ΔBamHI] or pEH3-Hbp[Δβ-cleav/ΔBamHI] thus forming expressionvectors corresponding to the vectors shown in FIGS. 3-10. FIG. 23 showsschematic representations of EspC-derivative constructs used in theexamples. For SEQ ID NO:s of the constructs, see table 2.

TABLE 2 Constructs used in this study Protein DNA Name SEQ ID NO SEQ IDNO Hbp(wild-type) 1 48 Hbp(Δβ-cleav) 2 49 HbpSS 3 50 Hbp(Δd1) (=HbpSL) 451 HbpD(Δd1) (=HbpDL) 5 52 Hbp(Δd2) 6 53 HbpD(Δd2) (=HbpDD2) 7 54Hbp(Δd3) 8 55 Hbp(Δd4) 9 56 Hbp(Δd5) 10 57 Hbp(d4ins) 11 58 HbpSS-ESAT612 59 Hbp(Δd1)-ESAT6 (=HbpSL-ESAT6) 13 60 HbpD(Δd1)-ESAT6 (=HbpDL-ESAT6)14 61 Hbp(Δd2)-ESAT6 15 62 HbpD(Δd2)-ESAT6 (=HbpDD2-ESAT6) 16 63Hbp(Δd3)-ESAT6 17 64 Hbp(Δd4)-ESAT6 18 65 Hbp(Δd5)-ESAT6 19 66Hbp(d4ins)-ESAT6 20 67 Hbp(βins)-ESAT6 21 68 Hbp(Δd3)-Rv2660c 22 69Hbp(Δd4)-Rv2660c 23 70 Hbp(Δd5)-Rv2660c 24 71 Hbp(Δd1)-TB10.4 25 72Hbp(Δd2)-TB10.3 26 73 Hbp(Δd1)-hEGF(0ss) 27 74 Hbp-Ag85B_([N+C]) 28 75HbpD-Ag85B_([N+C]) 29 76 Hbp-Ag85B_([C+N]) 30 77 HbpD-Ag85B_([C+N]) 3178 Hbp-Ag85B_([C+N])-ESAT6 32 79 HbpD-Ag85B_([C+N])-ESAT6 33 80Hbp-Ag85B_([C+N])-ESAT6-Rv2660c 34 81 HbpD-Ag85B_([C+N]) -ESAT6-Rv2660c35 82 EspC(wild-type) 36 83 EspC(Δd1) 37 84 EspC(Δd1)-ESAT6 38 85

Hbp(wild-type) is synthesized as a 1377 amino acid (aa) precursor thatis organized in three domains: (i) an N-terminal cleavable signalsequence (ss; aa 1-52), (ii) a passenger domain (aa 53-1100) and (iii)an outer membrane integrated C-terminal translocator domain (β-domain;aa 1101-1377). Domain 1 (d1), domain 2 (d2), domain 3 (d3), domain 4(d4), domain 5 (d5) and the autochaperone domain (ac) of the passengerdomain are indicated. “FL” denotes flexible linker. The remainder of thepassenger domain, including the beta stem domain is colored black. Afterpassage of the outer membrane the passenger is cleaved from thetranslocator domain via an autocatalytic mechanism that involveshydrolysis of the peptide bond between Asn¹¹⁰⁰ and Asn¹¹⁰¹ of the Hbpprecursor. Numbers displayed above the diagrams correspond to the aminoacid positions of the original Hbp(wild-type) precursor, calculated fromthe n-terminus.

“E-6” indicates ESAT6. “26” indicates Rv2660c. “10.3” indicates TB10.3and “10.4” indicates TB10.4”. “EGF” indicates hEGF(0ss). “85[N]”indicates Ag85B_([N′]) and “85[C]” indicates Ag85B_([C′]).

Hbp(Δβ-cleav) represents a mutant of Hbp(wild-type) of which thepassenger cannot be cleaved from the translocator domain due todisruption of the cleavage site (black cross) by substitution of Asn¹¹⁰⁰and Asn¹¹⁰¹ by a Gly and a Ser residue, respectively.

HbpSS represents a mutant of Hbp in which the vast majority of thepassenger, except the autochaperone domain has been substituted by aflexible linker (FL) hat allows insertion of heterologous proteinsequences.

Hbp(Δd1), Hbp(Δd2), Hbp(Δd3), Hbp(Δd4) and Hbp(Δd5) represent mutants ofHbp in which domain 1, 2, 3, 4 and 5, respectively, of the passenger hasbeen substituted by a flexible linker. Hbp(Δd1) is the same as HbpSL.

HbpD(Δd1) and HbpD(Δd2) are identical to Hbp(Δd1) and Hbp(Δd2),respectively, except that the cleavage site between the passenger andthe translocator domain was disrupted as described for Hbp(Δβ-cleav).HbpD(Δd1) has also been named HbpDL and HbpD(Δd2) has been named HppDD2.

Hbp(d4ins) is a mutant of Hbp in which residues 760-764—located indomain 4—have been substituted by a flexible linker.

HbpSS-ESAT6, Hbp(Δd1)-ESAT6, HbpD(Δd1)-ESAT6, Hbp(Δd2)-ESAT6,HbpD(Δd2)-ESAT6, Hbp(Δd3)-ESAT6, Hbp(Δd4)-ESAT6, Hbp(Δd5)-ESAT6 andHbp(d4ins)-ESAT6 are derivatives of HbpSS, Hbp(Δd1), HbpD(Δd1),Hbp(Δd2), HbpD(Δd2), Hbp(Δd3), Hbp(Δd4), Hbp(Δd5) and Hbp(d4ins),respectively. In these derivatives, an amino acid sequence correspondingto the ESAT6 the protein of Mycobacterium tuberculosis was inserted intothe flexible linker, leaving short flexible spacers comprising Gly andSer residues between the natural Hbp sequence and the N′ and C′ terminusof ESAT6.

Hbp(βins)-ESAT6 is a mutant of Hbp in which an amino acid sequencecorresponding to ESAT6 and short N′ and C′ flanking, flexible spacershas been inserted in a β-strand forming sequence of the Hbp passengerdomain: between residues 771 and 772.

Hbp(Δd3)-Rv2660c, Hbp(Δd4)-Rv2660c and Hbp(Δd5)-Rv2660c are derivativesof Hbp(Δd3), Hbp(Δd4) and Hbp(Δd5), respectively. In these derivatives,an amino acid sequence corresponding to the protein Rv2660c ofMycobacterium tuberculosis was inserted into the flexible linker asdescribed for Hbp(Δd3), Hbp(Δd4) and Hbp(Δd5) respectively.

Hbp(Δd1)-TB10.4 is a derivative of Hbp(Δd1) in which an amino acidsequence corresponding to the protein TB10.4 of Mycobacteriumtuberculosis was inserted into the flexible linker as described forHbp(Δd1).

Hbp(Δd2)-TB10.3 is a derivative of Hbp(Δd2) in which an amino acidsequence corresponding to the protein TB10.3 of Mycobacteriumtuberculosis was inserted into the flexible linker as described forHbp(Δd2).

Hbp(Δd1)-hEGF(0ss) is a derivative of Hbp(Δd1) in which an amino acidsequence corresponding to a cysteineless mutant of the protein hEGF ofHomo sapiens was inserted into the flexible linker as described forHbp(Δd1).

Hbp-Ag85B_([N+C]) is a mutant of Hbp in which an amino acid sequencecorresponding to residues 1-126 of the mature region of the proteinAg85B from Mycobacterium tuberculosis (Ag85B_([N′])) was inserted into aflexible linker that was located as described for Hbp(Δd1). In addition,an amino acid sequence corresponding to residues 118-285 of the matureregion of the protein Ag85B (Ag85B_([C′])) was inserted into a flexiblelinker that was located as described for Hbp(Δd2).

Hbp-Ag85B_([C+N]) is a mutant of Hbp in which an amino acid sequencecorresponding to residues 1-126 of the mature region of the proteinAg85B from Mycobacterium tuberculosis (Ag85B_([N′])) was inserted into aflexible linker that was located as described for Hbp(Δd2). In addition,an amino acid sequence corresponding to residues 118-285 of the e matureregion of the protein Ag85B (Ag85B_([C′])) was inserted into a flexiblelinker that was located as described for Hbp(Δd1).

Hbp-Ag85B_([C+N])-ESAT6 is derivative of Hbp-Ag85B_([C+N]) in which anamino acid sequence corresponding to ESAT6 was inserted into a flexiblelinker that was located as described for Hbp(d4ins). Thus Ag85B_([C′])is inserted at d1, Ag85B_([N′]) inserted at d2 and ESAT6 inserted at d4.

Hbp-Ag85B_([C+N])-ESAT6-Rv2660c is a derivative ofHbp-Ag85B_([C+N])-ESAT6 in which an amino acid sequence corresponding toRv2660c was inserted into a flexible linker that was located asdescribed for Hbp(Δd5). Thus Ag85B_([C′]) is inserted at d1,Ag85B_([N′]) inserted at d2, ESAT6 inserted at d4 and Rv2660c insertedat d5.

HbpD-Ag85B_([C+N]), HbpD-Ag85B_([C+N])-ESAT6 andHbpD-Ag85B_([C+N])-ESAT6-Rv2660c are derivatives of Hbp-Ag85B_([C+N]),Hbp-Ag85B_([C+N])-ESAT6 and Hbp-Ag85B_([C+N])-ESAT6-Rv2660c,respectively, except that the cleavage site between the passenger andthe translocator domain was disrupted as described for Hbp(Δβ-cleav).

General Procedures

SDS-PAGE was performed using 4-12% NuPAGE Bis-Tris gels (Invitrogen)with a MES-SDS running buffer. Alternatively, SDS-PAGE was performedusing 10%, 4-15% or ‘any-kD’ Biorad mini-Protean TGX gels, or standard10% SDS-PAGE gels. Before subjection to SDS-PAGE, protein samples weredissolved in SDS-PAGE sample buffer (63 mM TrisHcl pH 6.8, 2% w/v SDS,10% glycerol, 0.01% w/v bromophenol blue, 41 mM DTT) and boiled for 5min. Gels were stained with Coomassie Brilliant Blue G-250 and capturedusing a Molecular Imager GS-800 Calibrated Densitometer (Biorad).Alternatively, gels were subjected to Western blotting. Whereappropriate, Western blots were incubated with rabbit polyclonalantibodies directed against either the Hbp passenger domain, the Hbptranslocator domain, or the outer membrane protein OmpA. Alternatively,Western blots were incubated with mouse monoclonal antibodies directedagainst Mycobacterium tuberculosis ESAT6 or Ag85B, or with a ratpolyclonal antiserum directed against Rv2660c. Subsequently, Westernblots were incubated with horse-radish peroxidase (HRP) conjugated goatanti-rabbit antibodies (Rockland Immunochemicals), HRP-conjugated rabbitanti-mouse antibodies or HRP-conjugated rabbit anti-rat antibodies,where appropriate. Western blots were developed using chemiluminescentLumiLight Western blotting substrate (Roche). Chemiluminescent signalswere detected and digitalized using a ChemiDoc XRS+ Molecular Imager(BioRad).

Example 1 Expression and Biogenesis of Hbp Secretion and DisplayConstructs (FIG. 12)

This example illustrates proper expression and biogenesis of Hbpconstructs designed for the secretion or display of heterologous aminoacid sequences. Constructs carrying an intact cleavage site between thepassenger and the translocator domain (HbpSS and HbpSL) are properlyprocessed yielding translocator domains that are integrated into theouter membrane and passengers that are secreted into the medium.Expression of constructs carrying a disrupted cleavage site between thepassenger and the translocator domain (HbpDL and HbpDD2) yieldpassengers that remain covalently attached to the translocator domainand, hence, cell-associated.

Expression and secretion of Hbp, Hbp(Δβ-cleav), HbpSS, HbpSL (also namedHbp(Δd1)), HbpDL (also named HbpD(Δd1)) and HbpDD2 (also namedHbpD(Δd2)). E. coli MC1061 cells harboring the constructs cloned intothe expression vector pEH3 from overnight cultures were subcultured infresh medium and their growth was continued. When cultures reached earlylog phase (OD₆₆₀≈0.3), expression of Hbp(derivatives) was induced with 1mM of IPTG. Samples were collected from the cultures 2 h after inductionand cells (c) and spent medium (m) were separated by low speedcentrifugation. Cells were directly solubilized in SDS-PAGE samplebuffer whereas medium samples were subjected to TCA precipitation first.Samples corresponding to 0.03 OD₆₆₀ units of cells were analyzed bySDS-PAGE and Coomassie staining (A). Samples corresponding to 0.003OD₆₆₀ units of cells were analyzed by SDS-PAGE and Western blottingusing either polyclonal antibodies directed against the full-length Hbppassenger domain (B) or polyclonal antibodies directed against anN-terminal epitope of the Hbp translocator domain (C). Molecular mass(kDa) markers are indicated at the left side of the panels. Theprocessed passenger domains (>), processed translocator domains (#) andnon-processed pro-forms comprising both a passenger and translocatordomain (*) of the constructs are indicated.

Example 2 Expression and Biogenesis of Hbp Secretion Constructs Carryinga Heterologous Protein (FIG. 13)

This example illustrates that an heterologous protein ESAT6 isefficiently transported to the extracellular environment (culturemedium) via the Hbp autotransporter system when fused to the Hbppassenger at the position of domain 1 of the passenger (HbpSL-ESAT6).This example also shows that for the secretion of heterologous proteinsit is necessary to keep the beta stem of the Hbp passenger domainintact. This follows from the observation that fusion of ESAT6 to an Hbpconstruct of which the passenger has been N′ truncated up to theautochaperone domain (HbpSS-ESAT6) does not result in detectable amountsof ESAT6 in the culture medium.

Expression and secretion of Hbp, HbpSS, HbpSS-ESAT6, HbpSL (Hbp(Δd1))and HbpSL-ESAT6 (also named Hbp(Δd1)-ESAT6). E. coli MC1061 cellsharbouring the constructs cloned into the expression vector pEH3 or anempty vector (lane 1) from overnight cultures were subcultured in freshmedium and their growth was continued. When cultures reached early logphase (OD₆₀₀≈0.3), expression of Hbp(derivatives) was induced with 1 mMof IPTG. Samples were collected from the cultures 2 h after inductionand cells (c) and spent medium (m) were separated by low speedcentrifugation. Cells were directly solubilized SDS-PAGE sample bufferwhereas medium samples were subjected to TCA precipitation first.Samples corresponding to 0.03 OD₆₆₀ units of cells were analyzed bySDS-PAGE and Coomassie staining (A). Samples corresponding to 0.003OD₆₆₀ units of cells were analyzed by SDS-PAGE and Western blottingusing either polyclonal antibodies directed against an N-terminalepitope of the Hbp translocator domain (B), polyclonal antibodiesdirected against the full-length Hbp passenger domain (C) or monoclonalantibodies against the 10 kDa Mycobacterium tuberculosis protein ESAT6(E6) (D). Molecular mass (kDa) markers are indicated at the left side ofthe panels. The processed passenger domains (>), processed translocatordomains (#) and non-processed pro-forms comprising both a passenger andtranslocator domain (*) of the constructs are indicated.

Example 3 Expression and Biogenesis of Hbp Display Constructs Carrying aHeterologous Protein (FIG. 14)

This example illustrates that an heterologous protein ESAT6 is stablyexpressed when fused to the Hbp passenger at the position of domain 1(HbpDL-ESAT6) or domain 2 (HbpDD2-ESAT6) in an Hbp derivative carrying adisrupted cleavage site between the passenger and the translocatordomain.

Expression and secretion of Hbp(Δβ-cleav), HbpDL (HbpD(Δd1)),HbpDL-ESAT6 (Hbp(Δd1)-ESAT6), HbpDD2 (HbpD(Δd2) and HbpDD2-ESAT6(HbpD(Δd2)-ESAT6). E. coli MC1061 cells harbouring the constructs clonedinto the expression vector pEH3 from overnight cultures were subculturedin fresh medium and their growth was continued. When cultures reachedearly log phase (OD₆₆₀≈3), expression of Hbp-derivatives was inducedwith 1 mM of IPTG. After 2 hours of induction cells were collected bylow speed centrifugation and solubilized in SDS-PAGE sample buffer.Samples corresponding to 0.03 OD₆₆₀ units of cells were analyzed bySDS-PAGE and Coomassie staining (A). Samples corresponding to 0.003OD₆₆₀ units of cells were analyzed by SOS-PAGE and Western blottingusing either polyclonal antibodies directed against an N-terminalepitope of the Hbp translocator domain (B), polyclonal antibodiesdirected against the full-length Hbp passenger domain (C) or monoclonalantibodies against the 10 kDa Mycobacterium tuberculosis protein ESAT6(E6) (D). Molecular mass (kDa) markers are indicated at the left side ofthe panels. The non-processed pro-forms comprising both a passenger andtranslocator domain (*) of the constructs are indicated.

Example 4

Proteinase k Accessibility of Hbp-ESAT6 Fusions Displayed at the CellSurface (FIG. 15)

This example illustrates that the passengers of HbpDL and HbpDD2carrying ESAT6 are accessible to and, hence, degraded by proteinase kadded to intact cells, indicating that they are exposed to the cellsurface.

Proteinase k accessibility of Hbp(Δβ-cleav), HbpDL (HbpD(Δd1)),HbpDL-ESAT6 (HbpD(Δd1)-ESAT6), HbpDD2 (HbpD(Δd2)) and HbpDD2-ESAT6(HbpD(Δd2)-ESAT6). E. coli MC1061 cells harbouring the constructs clonedinto the expression vector pEH3 from overnight cultures were subculturedin fresh medium and their growth was continued. When cultures reachedearly log phase (OD₆₆₀≈0.3), expression of Hbp-derivatives was inducedwith 1 mM of IPTG. Cells were collected from the cultures 2 h afterinduction by low speed centrifugation and resuspended in 50 mM Tris-HCl,PH 7.4, containing 1 mM CaCl. In the case of Hbp(Δβ-cleav) and HbpDL,half of the cells were lysed by sonication on ice using a tip sonicator(Branson Sonifier 250). Subsequently, all samples were incubated withproteinase k (pk)(100 μg/ml) at 37° C. for 1 hour. The reaction wasstopped by addition of 0.1 mM phenylmethylsulfonyl fluoride (PMSF) andincubation on ice for 5 min. Samples were subjected to TCA precipitationbefore solubilization in SDS-PAGE sample buffer. To monitor theaccessibility of Hbp constructs displayed on intact cells to proteinasek, samples corresponding to 0.03 OD₆₆₀ units of cells were analyzed bySDS-PAGE and Coomassie staining (A). As a control, samples correspondingto 0.003 OD₆₆₀ units of cells were analyzed by SDS-PAGE and Westernblotting using polyclonal antibodies directed against the outer membraneprotein OmpA which is naturally inaccessible to proteinase k unlesscells are lysed by e.g. sonication (son)(B). An OmpA degradation productthat emerges upon proteinase k treatment is indicated (x). Molecularmass (kDa) markers are indicated at the left side of the panels. Thenon-processed pro-forms comprising both a passenger and translocatordomain (*) of the constructs are indicated. The position of proteinase K(pk) is indicated at the right hand side of the panels.

Example 5 Display of ESAT6 at the Cell Surface (FIG. 16)

This example illustrates that the heterologous protein ESAT6 fused tothe passenger of HbpDL or HbpDD2 is accessible to specific antibodiesadded to intact cells, indicating efficient display of ESAT6 at thecell-surface.

Surface display analysis of Hbp(Δβ-cleav), HbpDL (HbpD(Δd1)),HbpDL-ESAT6 (HbpD(Δd1)-ESAT6), HbpDD2 (HbpD(Δd2)) and HbpDD2-ESAT6(HbpD(Δd2)-ESAT6) and the secretion incompetent Hbp(ssTorA) andHbp(OMPLA). Hbp(ssTorA) is a mutant of Hbp which has its native signalpeptide replaced by the signal peptide of the protein TorA. Because theTorA signal peptide does not target Hbp to the Sec translocon, notranslocation across the inner membrane takes place and Hbp remains inthe cytoplasm. Hbp(OMPLA) is a mutant of Hbp which has its nativetranslocator domain replaced by the outer membrane protein OMPLA. OMPLAdoes target the Hbp passenger to the outer membrane but does not mediateits translocation across the outer membrane. Hence, the Hbp passengerremains orientated towards the periplasm and not to the extracellularmilieu.

E. coli MC1061 cells harbouring the constructs cloned into theexpression vector pEH3, or an empty vector (EV), from overnight cultureswere subcultured in fresh medium and their growth was continued. Whencultures reached early log phase (OD₆₆₀≈0.3), expression ofHbp-derivatives was induced with 1 mM of IPTG. Cells were collected 1hour after induction by low speed centrifugation, washed in icecold 50mM Tris-HCl, PH 7.4, and eventually resuspended in ice-cold 50 mMTris-HCl, PH 7.4 and left on ice. Half of each sample was subjected totip sonication on ice (Branson Sonifier 250) to lyse the cells, whereasthe cells of the other half were left intact. Subsequently, a five-folddilution range of each sample was prepared in icecold 50 mM Tris-HCl, pH7.4. Dilutions of each sample were applied on presoaked nitrocellulosemembranes using a vacuum manifold based Bio-Dot apparatus (Biorad).Membranes were blocked upon incubation in a 5% skimmed milk solution inTBS for 20 min. To detect surface exposure of the passenger ofHbp-derivatives, membranes were incubated with rabbit polyclonalantibodies directed against the Hbp passenger in TBS for 1 h, washed 3times with TBS, incubated with HRP conjugated goat anti-rabbitantibodies in TBS for 45 min, washed 3 times with TBS and developedusing di-octylsodiumsulphosuccinate (DONS) staining (A). This confirmedsurface-exposure of the passengers of Hbp(Δβ-cleav), HbpDL, HbpDL-ESAT6,HbpDD2 and HbpDD2-ESAT6 on whole cells as opposed to the passengers ofsecretion-incompetent mutants Hbp(ssTorA) and Hbp(OMPLA) the expressionof which was apparent from the corresponding sonicated samples.

To demonstrate display of ESAT6 by HbpDL-ESAT6 and HbpDD2-ESAT6 on wholecells the same procedure was followed as under A except that mousemonoclonal antibodies directed against ESAT6 were used and HRPconjugated rabbit anti-mouse antibodies (B). As a control, it wasdemonstrated that a periplasmic protein OppA could not be efficientlydetected on whole cells as opposed to the sonicated samples. For this,the same procedure was used as described under A except that a rabbitpolyclonal antiserum against OppA was used (C). At the left hand side ofthe panels the amount of material (in OD₆₆₀ units) applied is indicated.

Example 6 Biogenesis of Hbp Upon (Partial) Deletion of Side Domains(FIG. 17)

This example illustrates successful secretion of Hbp upon replacement ofeither of the side domains 1 to 5 by a flexible amino acid linkersequence (Δd1-Δd5). Furthermore, successful secretion of an insertionmutant (d4ins) is shown in which only 4 amino acids of domain 4 arereplaced by a flexible linker.

Expression and secretion of Hbp, Hbp(Δd1), Hbp(Δd2), Hbp(Δd3), Hbp(Δd4),Hbp(Δd5) and Hbp(d4ins). E. coli MC1061 cells harbouring the constructscloned into the expression vector pEH3 or an empty vector (−) fromovernight cultures were subcultured in fresh medium and their growth wascontinued. When cultures reached early log phase (OD₆₆₀≈0.3), expressionof Hbp(derivatives) was induced with 1 mM of IPTG. Samples werecollected from the cultures 2 h after induction and cells (c) and spentmedium (m) were separated by low speed centrifugation. Cells weredirectly solubilized SDS-PAGE sample buffer whereas medium samples weresubjected to TCA precipitation first. Samples corresponding to 0.03OD₆₆₀ units of cells were analyzed by SDS-PAGE and Coomassie staining.

Proper secretion follows from the appearance of cleaved passenger domain(>) in the cell fraction (c) and culture medium (m), and cleavedtranslocator domain (β) in the cell fraction, similar to wild-type Hbp(wt) (FIG. 17). Molecular mass (kDa) markers are indicated at the leftside of the panel.

Example 7 Secretion of ESAT6 Fused to the Position of Either of theDomains d1 to d5 (FIG. 18)

This example illustrates efficient secretion of the Mycobacteriumtuberculosis antigen ESAT6 upon fusion to the Hbp passenger domain atthe position of either of the domains d1 to d5, or insertion into domain4 (d4ins).

Expression and secretion Hbp(Δd1)-ESAT6, Hbp(Δd2)-ESAT6, Hbp(Δd3)-ESAT6,Hbp(Δd4)-ESAT6, Hbp(Δd5)-ESAT6 and Hbp(d4ins)-ESAT6. (A) E. coli MC1061cells harbouring the constructs cloned into the expression vector pEH3were grown, induced and analyzed as described under Example 6. (B)Samples from A corresponding to 0.003 OD₆₆₀ units of cells were analyzedby Western blotting using monoclonal antibodies directed against ESAT6.

Proper secretion follows from the appearance of cleaved passenger domain(>) in the cell fraction (c) and culture medium (m), and cleavedtranslocator domain (β) in the cell fraction (FIG. 18 A). The presenceof ESAT6 in the respective passenger domains is confirmed by Westernblotting using ESAT6 specific antibodies (FIG. 18 B). Molecular mass(kDa) markers are indicated at the left side of the panels.

Example 8 Secretion of TB10.3 and TB10.4 Upon Replacement of Domain d1or d2 (FIG. 19)

This example illustrates efficient secretion of the Mycobacteriumtuberculosis proteins TB10.3 and TB10.4 upon replacement of domain d2and domain d1 of the Hbp passenger, respectively.

Expression and secre ion of Hbp(Δd1)-TB10.4 and Hbp(Δd2)-TB10.3. E. coliTOP10F′ cells harbouring the constructs cloned into the expressionvector pEH3, or carrying a non-expressing plasmid (−), were grown andinduced as described under Example 6. Samples were withdrawn from thecultures 2 h after induction. Subsequently, cells were isolated bycentrifugation, solubilized in SDS-PAGE sample buffer and analyzed byCoomassie stained SDS-PAGE.

Proper secretion follows from the appearance of cleaved passenger domain(>) in the cell fraction (c) and culture medium (m), and cleavedtranslocator domain (β) in the cell fraction (FIG. 19). Molecular mass(kDa) markers are indicated at the left side of the panel.

Example 9 Secretion of Rv2660c Fused to the Position of Either of theDomains d3, d4 or d5 (FIG. 20)

This example illustrates efficient secretion of the Mycobacteriumtuberculosis antigen Rv2660c upon fusion to the Hbp passenger domain atthe position of domain 3, domain 4 or domain 5.

Expression and secretion of Hbp(Δd3)/Rv2660c, Hbp(Δd4)/Rv2660c,Hbp(Δd5)/Rv2660c. E. coli MC1061 cells harbouring the constructs clonedinto the expression vector pEH3 were grown, induced and analyzed asdescribed under Example 6.

Proper secretion follows from the appearance of cleaved passenger domain(>) in the cell fraction (c) and culture medium (m), and cleavedtranslocator domain (β) in the cell fraction (FIG. 20). Molecular mass(kDa) markers are indicated at the left side of the panel.

Example 10 Secretion of Cysteinless hEGF (FIG. 21)

This example illustrates efficient secretion of a cysteineless (0ss)version of the Homo sapiens protein hEGF (EGF) upon fusion to the Hbppassenger domain at the position of domain 1.

Expression and secretion of Hbp(wild-type), Hbp(Δd1) andHbp(Δd1)-hEGF(0ss). E. coli MC1061 cells harbouring the constructscloned into the expression vector pEH3 were grown overnight in M9 mediumsupplemented with glucose (0.4%), chloramphenicol (20 μg/ml) andStreptomycin (30 μg/ml) at 37° C. Next morning cells were subcultured infresh medium and their growth was continued. When cultures reached earlylog phase (OD₆₆₀≈0.3), expression of Hbp(derivatives) was induced with 1mM of IPTG. Samples were collected from the cultures 3 h after inductionand cells (c) and spent medium (m) were separated by low speedcentrifugation. Cells were directly solubilized SDS-PAGE sample bufferwhereas medium samples were subjected to TCA precipitation first. Cellsamples corresponding to 0.05 OD₆₆₀ units and medium samplescorresponding to 0.1 OD₆₆₀ units were analyzed by SDS-PAGE and Coomassiestaining.

Proper secretion follows from the appearance of cleaved passenger domain(>) in the cell fraction (c) and culture medium (m), and cleavedtranslocator domain (β) in the cell fraction (FIG. 21). Molecular mass(kDa) markers are indicated at the left side of the panel.

Example 11 Impaired Secretion of ESAT6 Upon Insertion into β-StemForming Sequence (FIG. 22)

This example illustrates that insertion of ESAT6 in the β-stem formingsequence of the Hbp passenger domain (βins-ESAT6) yields inefficientsecretion as compared to replacement of side domain d4 by ESAT6(Δd4-ESAT6). Of note, the ESAT6 fusion sites in the respectiveconstructs are only 5 amino acid residues apart.

Expression and secretion of Hbp(wild-type), Hbp(Δd4)-ESAT6 andHbp(βins)-ESAT6. E. coli MC1061 cells harbouring the constructs clonedinto the expression vector pEH3 were grown and induced as describedunder Example 6. Samples were withdrawn from the cultures 2 h afterinduction. Subsequently, cells were isolated by centrifugation,solubilized in SDS-PAGE sample buffer and analyzed by Coomassie stainedSDS-PAGE. Molecular mass (kDa) markers are indicated at the left side ofthe panels. Cleaved Hbp passenger species are indicated (>).

Example 12 Secretion of ESAT6 Upon Fusion to an AlternativeAutotransporter (FIG. 23-24)

This example illustrates that the methodology used to secreteheterologous proteins via the Hbp secretion system is applicable to analternative autotransporter (AT); EspC.

FIG. 23 A shows a model of the EspC passenger domain structure. Thestructure was predicted in silico using the M4T homology modeling method(Rykunov et al 2009 J Struct Funct Genomics 10: 95-99). The primaryamino acid sequence corresponding to the EspC passenger domain (residues54-1028 of the protein with accession number Q9EZE7) was used as input.Side domains protruding from the β stem were identified. Domain 1 (d1)is in light grey. The remainder of the passenger domain, including thebeta stem domain is colored black. Domain d1 is suitable for replacementby a POI.

FIG. 23 B shows schematic representations of EspC derivatives used inthe examples. EspC(wild-type) is synthesized as a 1306 amino acid (aa)precursor that is organized in three domains: (i) an N-terminalcleavable signal sequence (ss; aa 1-53), (ii) a passenger domain (aa54-1028) and (iii) an outer membrane integrated C-terminal translocatordomain (β-domain; aa 1029-1306). The predicted domain 1 (d1) isindicated. The remainder of the passenger domain, including the betastem domain, is colored black. “FL” denotes flexible linker. “E-6”indicates ESAT6. After passage of the outer membrane, the passenger iscleaved from the translocator domain (β-domain) via an autocatalyticmechanism that involves hydrolysis of the peptide bond between Asn¹⁰²⁸and Asn¹⁰²⁹ of the EspC precursor. Numbers displayed above the diagramscorrespond to the amino acid positions of the original EspC(wild-type)precursor, calculated from the n-terminus.

Expression and secretion of EspC, EspC(Δd1), and EspC(Δd1)-ESAT6. (FIG.24 A) E. coli MC1061 cells harbouring the constructs cloned into theexpression vector pEH3 were grown, induced and analyzed as describedunder Example 6. (FIG. 24 B) Samples from A corresponding to 0.003 OD₆₆₀units of cells were analyzed by Western blotting using monoclonalantibodies against ESAT6.

Here, it is shown that upon replacement of the predicted dom1 of thepassenger domain by a flexible amino acid sequence (Δd1) secretion ofthe EspC passenger proceeds with the same efficiency as wild-type EspC(wt). Furthermore, this example illustrates that ESAT6 can beefficiently secreted upon fusion to the EspC passenger at the positionof the predicted domain d1 (Δd1/ESAT6). Proper secretion follows fromthe appearance of cleaved passenger domain (>) in the medium fraction(m) and cleaved, and cleaved translocator domain in the cell fraction(x) (FIG. 24 A). The presence of ESAT6 in the EspC(Δd1/ESAT6) passengerdomain is confirmed by Western blotting using ESAT6 specific antibodies(FIG. 24 B). Molecular mass (kDa) markers are indicated at the left sideof the panels.

Example 13 Secretion of Split Ag85B and ESAT6 (FIG. 25)

This example illustrates the simultaneous secretion of the amino acidstretches Ag85B[N′] and Ag85B[C′], roughly corresponding to the N′ andC′ terminal half of the mature region of the Mycobacterium tuberculosisantigen Ag85B, respectively. Secretion of the two moieties was achievedupon translational fusion of Ag85B[N′] to the Hbp passenger at theposition of domain 1 and Ag85B[C′] at the position of domain 2 of thesame Hbp passenger molecule (85_([N+C])). Alternatively, Ag85B[C′] wasfused at the position of domain 1 and Ag85B[N′] at the position ofdomain 2 (85_([N+C])).

In addition, this example illustrates the simultaneous secretion ofAg85B[N′], Ag85B[C′] and the mycobacterial antigen ESAT6. This wasachieved upon fusion of Ag85B[N′] at the position of domain 1, Ag85B[C′]at the position of domain 2 and insertion of ESAT6 into domain 4 of thesame Hbp passenger molecule (85_([N+C])/E6). Alternatively, Ag85B[C′]was fused at the position of domain 1, Ag85B[N′] fused at the positionof domain 2 and ESAT6 inserted into domain 4 (85_([C+N])/E6).

Expression, secretion and proteinase K accessibility of Hbp,Hbp(Δβ-cleav), Hbp-Ag85B_([N+C]), Hbp-Ag85B_([C+N]),Hbp-Ag85B_([N+C])/ESAT6, Hbp-Ag85B_([C+N])/ESAT6. (FIG. 25 A) E. coliTOP10F′ cells harbouring the constructs cloned into the expressionvector pEH3 or an empty vector (−) from overnight cultures were grown,induced and analyzed as described under Example 6. (FIG. 25 B) Cells asgrown and induced under A were resuspended in 50 mM Tris-HCl, PH 7.4,containing 1 mM CaCl. Subsequently, samples were incubated at 37° C. for1 hour with (+) or without (−) proteinase k (pk) (100 μg/ml). Thereaction was stopped by addition of 0.1 mM phenylmethylsulfonyl fluoride(PMSF) and incubation on ice for 5 min. Samples were subjected to TCAprecipitation before solubilization in SDS-PAGE sample buffer andanalysis on Coomassie stained SDS-PAGE. Molecular mass (kDa) markers areindicated at the left side of the panels. Cleaved passengers (>) andtranslocator domains (β), the position of proteinase K (pk) isindicated.

Proper secretion follows from the appearance of cleaved passenger domain(>) and translocator domain (β) in the cell fraction (c), similar towild-type Hbp (A). As a control, no cleaved passenger and translocatordomain is observed for a non-cleavable, but translocation competentversion of Hbp (Δβ). To confirm their extracellular location,sensitivity of the passengers towards proteinase K added to intact cellsis shown (8).

Example 14 Simultaneous Secretion of Split Ag85B, ESAT6 and Rv2660 (FIG.26)

This example illustrates the simultaneous secretion of Ag85B[N′],Ag85B[C′], ESAT6 and the Mycobacterial antigen Rv2660c when fused to asingle Hbp passenger domain. Secretion the moieties was achieved upontranslational fusion of Ag85B[C′] to the Hbp passenger at the positionof domain 1, fusion of Ag85B[N′] at the position of domain 2, insertionof ESAT6 into domain 4, and fusion of Rv2660c at the position of domain5 (“85-E6-2660”). For comparison, the secretion of constructs onlycarrying Ag85B[C′] and Ag85B[N′] (“85”) or Ag85B[C′], Ag85B[N′] andESAT6 (“85-E6”) were analyzed in parallel.

Expression and secretion of Hbp-Ag85B_([C+N]), Hbp-Ag85B_([C+N])/ESAT6and Hbp-Ag85B_([C+N])/ESAT6/Rv2660c. E. coli MC1061 cells harbouring theconstructs cloned into the expression vector pEH3 were grown and inducedas described under Example 6.

Proper secretion follows from the appearance of cleaved passenger domain(>) and translocator domain (x) in the cell fraction (c) (FIG. 26).Molecular mass (kDa) markers are indicated at the left side of thepanel.

Example 15 Simultaneous Display of Split Ag85B, ESAT6 and Rv2660c (FIG.27)

This example illustrates the simultaneous display of Ag85B[N′],Ag85B[C′], ESAT6 and Rv2660c when fused to a single passenger domain ofHbp(Δβcleav), a non-cleavable, yet translocation competent version ofHbp. Display was achieved upon translational fusion of Ag85B[C′] to theHbp passenger at the position of domain 1, fusion of Ag85B[N′] at theposition of domain 2, insertion of ESAT6 into domain 4, and fusion ofRv2660c at the position of domain 5 (85-E6-2660). For comparison, thedisplay of constructs only carrying Ag85B[C′] and Ag85B[N′] (85), orAg85B[C′], Ag85B[N′] and ESAT6 (85-E6) were analyzed in parallel.Furthermore, display of Hbp(Δβcleav) (Δβ) was analyzed (FIG. 27 A).

Westen blotting using specific antibodies against Ag85B[C′], ESAT6 andRv2660c was carried out to confirm the presence of these moieties in therespective passenger domains where appropriate (FIG. 27 B).

To confirm translocation of the respective passenger domains across thecell envelope, and their display at the cell surface, sensitivity of thepassengers towards proteinase K added to intact cells is shown (FIG. 27C).

In FIG. 27 molecular mass (kDa) markers are indicated at the left sideof the panels. Non-cleaved pro-form Hbp species are indicated (*).

Expression and display of Hbp(Δβcleav), HbpD-Ag85B_([C+N]),HbpD-Ag85B_([C+N])/ESAT6 and HbpD-Ag85B_([C+N])/ESAT6/Rv2660c, (A) E.coli MC1061 cells harbouring the constructs cloned into the expressionvector pEH3 were grown and induced as described under Example 6. Sampleswere withdrawn from the cultures 2 h after induction. Subsequently,cells were isolated by centrifugation, solubilized in SDS-PAGE samplebuffer and analyzed by Coomassie stained SDS-PAGE. (B) Samples from Acorresponding to 0.003 OD₆₆₀ units of cells were analyzed by Westernblotting using either monoclonal antibodies directed against an epitopeof Ag85B_([C]), monoclonal antibodies directed against ESAT6 orpolyclonal antibodies directed against Rv2660c. (C) Cells as grown andinduced under A were resuspended in 50 mM Tris-HCl, PH 7.4, containing 1mM CaCl. Subsequently, samples were incubated at 0° C. for 30 min with(+) or without (−) proteinase k (pk) (100 μg/ml). The reaction wasstopped by addition of 0.1 mM phenylmethylsulfonyl fluoride (PMSF) andincubation on ice for 5 min. Samples were subjected to TCA precipitationbefore solubilization in SDS-PAGE sample buffer and analysis onCoomassie stained SDS-PAGE.

Example 16 Simultaneous Secretion of Ag85B, ESAT6 and Rv2660c by anAttenuated Salmonella Strain (FIG. 28)

This example illustrates the simultaneous secretion of Ag85B[N′],Ag85B[C′], ESAT6 and the Mycobacterial antigen Rv2660c, when fused to asingle Hbp passenger domain, by attenuated Salmonella typhimurium.Secretion the moieties was achieved upon translational fusion ofAg85B[C′] to the Hbp passenger at the position of domain 1, fusion ofAg85B[N′] at the position of domain 2, insertion of ESAT6 into domain 4,and fusion of Rv2660c at the position of domain 5 (85_([C+N])-E6-2660).Proper secretion follows from the appearance of cleaved passenger domain(>) in the medium fraction (m) (FIG. 28 A).

Westen blotting using specific antibodies against Ag85B[C′], ESAT6 andRv2660c was carried out to confirm the presence of these moieties in thesecreted passenger domain. Furthermore, Western blotting using anantiserum against the Hbp translocator domain (abarrel) confirmed theoccurrence of cleaved translocator domain in the cells (FIG. 288).

Expression and secretion of Hbp-Ag85B_([C+N])-ESAT6-Rv2660c byattenuated Salmonella typhimurium. (A) Salmonella typhimurium strainSL3261 (Hoiseth and Stocker 1981 Nature 291: 281-282) and a derivativecarrying a single copy of the gene encodingHbp-Ag85B_([C+N])-ESAT6-Rv2660c on the genome under control of aconstitutive lacUV5 promoter were grown overnight to saturation in LBmedium at 37° C. For construction of Salmonella strains see Example 21.Next morning, the cells were subcultured in fresh medium and theirgrowth was continued. Two hours after subculturing, samples werecollected from the cultures and cells (c) and spent medium (m) wereseparated by low speed centrifugation. Cells were directly solubilizedin SDS-PAGE sample buffer whereas medium samples were subjected to TCAprecipitation first. Samples corresponding to 0.03 OD₆₆₀ units of cellswere analyzed by SDS-PAGE and Coomassie staining. (8) Samples from Acorresponding to 0.003 OD₆₆₀ units of cells were analyzed by Westernblotting using either monoclonal antibodies directed against an epitopeof Ag85B_([C]), monoclonal antibodies directed against ESAT6, polyclonalantibodies directed against Rv2660c, or polyclonal antibodies directedagainst the Hbp translocator domain.

A background band of unknown identity (*) is indicated. Molecular mass(kDa) markers are indicated at the left side of the panels.

Example 17 Simultaneous Display of Ag85B, ESAT6 and Rv2660c by anAttenuated Salmonella Strain (FIG. 29)

This example illustrates the expression and simultaneous display ofAg85B[N′], Ag85B[C′], ESAT6 and Rv2660c at the cell surface ofattenuated Salmonella typhimurium, when fused to a single Hbp passengerdomain of passenger domain of Hbp(Δβcleav), a non-cleavable, yettranslocation competent version of Hbp(HbpD-Ag858_([C+N])-ESAT6-Rv2660c). Also shown is the surface display ofa single ESAT6 unit using the same strategy (HbpD-ESAT6).

Proper expression of the constructs follows from analysis by SDS-PAGEand Coomassie staining showing the appearance of protein bands with amolecular weight corresponding to the calculated molecular weight ofHbpD-Ag85B_([C+N])-ESAT6-Rv2660c and HbpD-ESAT6, respectively (*). As acontrol, these bands are not present in non-expressing control cells(−).

To confirm translocation of the respective passenger domains across thecell envelope and display at the cell surface, their sensitivity towardsproteinase K (pk) added to intact cells is shown.

Expression and display of HbpD-Ag85B_([C+N])-ESAT6-Rv2660c andHbpD-ESAT6 on the cell surface of attenuated Salmonella typhimurium.Salmonella typhimurium strain SL3261 (Hoiseth and Stocker 1981 Nature291: 281-282) (−) and derivative carrying either a single copy of thegene encoding HbpD-Ag85B_([C+N])-ESAT6-Rv2660c or the gene encodingHbpD-ESAT6 on the genome under control of a constitutive lacUV5promoter, were grown overnight to saturation in LB medium at 37° C. Forconstruction of Salmonella strains see Example 21. Next morning, thecells were subcultured in fresh medium and their growth was continued.Two hours and 30 min after subculturing, samples were collected from thecultures and cells were resuspended in 50 mM Tris-HCl, PH 7.4,containing 1 mM CaCl. Subsequently, samples were incubated at 37° C. for1 hour with (+) or without (−) proteinase k (pk) (100 μg/ml), Thereaction was stopped by addition of 0.1 mM phenylmethylsulfonyl fluoride(PMSF) and incubation on ice for 5 min. Samples were subjected to TCAprecipitation, solubilized in SDS-PAGE sample buffer and analyzed onCoomassie stained SDS-PAGE. The non-processed pro-forms of theconstructs (*), comprising both a passenger and translocator domain, areindicated. Molecular weight markers (kDa) are displayed at the righthand side of the panels.

Example 18 Simultaneous Display of Split Ag85B, ESAT6 and Rv2660c onOuter Membrane Vesicles (FIG. 30A-D)

This sample illustrates the simultaneous display of Ag85B[C′], Ag85B[N′]and ESAT6 on the surface of bacterial outer membrane vesicles (OMVs)upon fusion to a single passenger domain of Hbp(Δβcleav), anon-cleavable, yet translocation competent version of Hbp (85B-E6-2660).In addition, the combined display of Ag85B[C′], Ag85B[N′] and ESAT6(85B-E6), as well as a single ESAT6 unit (Δd1-E6) is shown. As acontrol, the display of HbpD(Δd1) not carying a heterologous partner(Δd1) was analyzed. To achieve display on OMVs, the fusion proteins wereexpressed in an E. coli strain carrying mutations in the tol-pal genesinducing a hyper-vesiculating phenotype.

Localization of the fusion proteins in OMVs is shown by theircolocalization with outer membrane porin proteins OmpA and OmpC in OMVisolates derived from filtrated and, hence, cell-free culture mediumfractions (FIGS. 30A and B). Successful display of the fusion proteinsat the surface of OMVs is shown by their sensitivity towards proteinaseK added to the OMVs externally (FIGS. 30C and D). To confirm theintegrity of the OMVs, it is shown that the proteinase K sensitiveintracellular domain of OmpA is not accessible, unless the OMVs aresolublized using the detergent triton x-100 (tx-100).

Expression and display of HbpD(Δd1), HbpD(Δd1)-ESAT6,HbpD-Ag85B_([C+N])-ESAT6 and Ag85B_([C+N])-ESAT6-Rv2660c on OMVs. (A) E.coli JC8031 cells (Barnadac et al 1998 Journal of Bacteriology 180:4872-4878) harbouring the constructs cloned into the expression vectorpEH3 or an empty vector (−) from overnight cultures were subcultured infresh medium and their growth was continued. When cultures reached earlylog phase (OD₆₆₀≈0.2), expression of Hbp(derivatives) was induced with 1mM of IPTG. Three hours after induction 50 ml culture samples werecentrifuged (5000 rpm, 4° C., 15 min) to separate the cells from themedium. Cells (Total cells) were solubilized in SDS-PAGE sample bufferwhereas the culture medium was subjected to centrifugation once more(5000 rpm, 4° C., 15 min). The resulting supernatant was filteredthrough 0.2 μm-pore-size filters and subjected to high-speedcentrifugation (45,000 rpm, 4° C., 1 h) using a Kontron TFT 70.38 rotor.The pellet fraction, containing the OMVs, was resuspended in PBS. Asample corresponding to 1 OD660 unit of cells was solubilized inSDS-PAGE sample buffer and analyzed by Coomassie stained SDS-PAGE inparallel to 0.02 OD660 units of Total cells. The outer membrane proteinsOmpA and OmpC, the identity of which was confirmed by Mass specanalysis, have been indicated at the left side of the panel. (B) Toconfirm the identity of the fusion proteins, samples prepared under Awere analyzed by Western blotting using a polyclonal antiserum againstthe Hbp translocator domain (αbarrel) and monoclonal antibodies againstESAT6. (C) Proteinase K treatment of OMVs. OMVs isolated under A wereresuspended in 50 mM Tris-HCl, PH 7.4, containing 1 mM CaCl. Sampleswere split into three equal aliquots, which were incubated with (+) orwithout (−) Proteinase K (pk) (100 μg/ml) as indicated. Prior toaddition of proteinase k, triton X-100 (tx-100) (1%) was added to one ofthe aliquots. All aliquots were incubated at 37° C. for 30 min, afterwhich the reaction was stopped by addition of 0.1 mMphenylmethylsulfonyl fluoride (PMSF) and incubation on ice for 5 min.Samples were subjected to TCA precipitation, solubilized in SDS-PAGEsample buffer. Samples corresponding to 1 OD660 unit of cells were byCoomassie stained SDS-PAGE. (D) To confirm the identity of the fusionproteins, samples prepared under C were analyzed by Western blottingusing a polyclonal antiserum against the Hbp translocator domain(abarrel) and monoclonal antibodies against ESAT6. The non-processedpro-forms of the constructs (*), comprising both a passenger andtranslocator domain, are indicated. Molecular weight markers (kDa) aredisplayed at the right hand side of the panels.

Example 19 Display of Split Ag85B and ESAT6 on Ghosts (FIG. 31A-C)

In the present Example 19 plasmid pLargeRhaLysisE was used to turn E.coli cells into ghosts using the herein described methodology. Theconstruction of pLargeRhaLysisE is described in Example 20.

This sample illustrates the simultaneous display of Ag85B_([C]),Ag85B_([N]) and ESAT6 on the surface of bacterial ghosts upon fusion toa single passenger domain of Hbp(Δβcleav). To achieve surface display onghosts, HbpD-Ag85B_([C+N]). ESAT6 was expressed in E. coli cellstransformed with a plasmid that carries the gene encoding thebacteriophage phiX174 lysis protein E under control of an induciblepromoter. Following expression of HbpD-Ag85B_([C+N]). ESAT6, expressionof the lysis protein E was induced, leading to the release of thecellular cytoplasmic content into the culture medium. This resulted inthe emergence of ‘empty’ bacterial cell envelopes (ghosts) displayingHbpD-Ag85B_([C+N]). ESAT6 at the surface.

Succesful lyis protein E mediated ghost formation is shown by a drop inapparent cell density (OD600) upon lyis protein E expression (FIG. 31A).Furthermore, it is shown that cytoplasmic marker proteins (SecB, DnaK)are released into the medium upon expression of lysis protein E and,hence, end up in the supernatant fraction after centrifugation. Incontrast, a periplasmic marker protein (SurA) and an integral membraneprotein (Lep) (mainly) localize to the centrifugation pellet containingthe bacterial cell envelopes (ghosts) (FIG. 31B). Correct localizationof HbpD-Ag85B_([C+N]). ESAT6 in the ghosts is shown by itscolocalization with Lep in the pellet fraction (FIG. 31B). Successfuldisplay of HbpD-Ag85B_([C+N]). ESAT6 at the surface of ghosts andcontrol cells is shown by its sensitivity towards proteinase K added tothe ghosts/cells externally (FIG. 31C).

Expression and display of HbpD-Ag85B_([C+N])-ESAT6 and the subsequentformation of ghosts. (A) E. coli MC4100 cells co-transformed with (i) apEH3_((p15a))-HbpD-Ag85B_([C+N])-ESAT6, and (ii) pLargeRhaLysisE,carrying the gene encoding lysis protein E from bacteriophage phiX174under control of an rhamnose inducible promoter, were grown in LB mediumat 30° C. When the culture reached an OD600 of 0.5, 0.4 mM of IPTG wasadded to induce the expression of HbpD-Ag85B_([C+N])-ESAT6 and theculture was split. One hour after addition of IPTG, 0.2% rhamnose wasadded to one half of the original culture to induce the expression oflysis protein E, whereas the other half of the culture was used as acontrol. The OD600 of the cultures was monitored over time. (B) Threehours after IPTG induction, the control cells (non-induced culture) andghosts (induced culture) grown under A were isolated by centrifugation.The supernatant containing the culture medium was isolated and itsprotein content was TCA precipitated. Cell/ghost pellets (P; cell/ghostpellet) and TCA precipitated material (S; supernatant) were thensolubilized in SDS-PAGE sample buffer and analyzed by SDS-PAGE andWestern blotting. The presence of HbpD-Ag85B_([C+N])-ESAT6 was detectedusing polyclonal antibodies directed against the Hbp passenger (pass.)and translocator domain (barrel), and monoclonal antibodies againstESAT6. The formation of ghosts was monitored using polyclonal antibodiesagainst the cytoplasmic proteins DnaK and SecB, the periplasmic proteinSurA and the integral membrane protein Lep. (C) Proteinase K treatmentof control cells and ghosts. Part of the cells and ghosts isolated under8 were resuspended in 50 mM Tris-HCl, PH 7.4, containing 1 mM CaCl.Proteinase K (100 pg/ml) was added to half of each sample (+) whereasthe other half was left untreated (−). Samples were incubated at 37° C.for 1 h, after which the reaction was stopped by addition of 0.1 mMphenylmethylsulfonyl fluoride (PMSF) and incubation on ice for 5 min.Samples were subjected to TCA precipitation, solubilized in SDS-PAGEsample buffer and analyzed by Western blotting using an antiserumagainst the Hbp passenger domain.

Example 20

The following example relates to construction of plasmidpLargeRhaLysisE. FIG. 32 shows a plasmid map of pLargeRhaLysisE used inExample 20. Table 3 and Table 4 below show, respectively, the primersequences and the constructs used in Example 20.

Plasmid pLargeRhaLysisE carries the gene encoding lysis protein E frombacteriophage phiX174 under control of a rhamnose inducible rhaBADpromoter. To construct pLargeRhaLysisE, plasmid pLarge was constructedfirst, which is based on pSB3398 (Wagner et al 2010 Proc Natl Acad SciUSA 107:17745-17750) and pRha67K.

Plasmid pRha67K is a derivative of pRha67 (Giacalone et al. 2006BioTechniques 40: 355-364) where the gene encoding the ampicillin markeris replaced from the start to the stop codon by the gene encoding thekanamycin marker from pET28(a+) (EMD Biosciences) using the USER cloningmethod (Bitinaite and Nichols 2009 Curr Protoc Mol Biol Chapter 3:Unit3.21; Nørholm 2010 BMC Biotechnol 10: 21). The gene encoding thekanamycin resistance marker of pET28(a+) was amplified using pET28(a+)as a template and the deoxyuracil (u) containing primers kanR and kanF.DNA encoding pRha67 without the gene encoding the ampicillin marker wasamplified using the deoxyuracil containing primers pRhakanF andpRhakanR. The PfuX7 polymerase was used to amplify DNA using deoxyuracilcontaining primers (Nørholm 2010 BMC Biotechnol 10: 21). Subsequently,USER Enzyme (New England Biolabs) was used according to the instructionsof the manufacturer for the construction of pRha67K.

Plasmid pLarge is a derivative of pRha67K where the rhamnose promoter(including regulatory elements), multiple cloning site and terminatorare replaced by the ones from pSB3398 (VVagner et al 2010 Proc Natl AcadSci USA 107:17745-17750) using the USER cloning method (Bitinaite andNichols 2009 Curr Protoc Mol Biol Chapter 3:Unit 3.21; Nørholm 2010 BMCBiotechnol 10: 21). The regulatory elements (rhaR, rhaS PrhaBAD), andthe multiple cloning site and transcriptional terminator rrnB wereamplified using pSB3398 as a template and the deoxyuracil containingprimers pSB3398 forward and pSB3398 reverse. pRha67K was used as atemplate to amplify the part of pRha67K covering the kanamycinresistance marker (including its promoter and terminator) and origin ofreplication. The deoxyuracil containing primers used were 67kF andpRhaR. The PfuX7 polymerase was used to amplify the DNA and,subsequently, USER Enzyme (New England Biolabs) was used according tothe instructions of the manufacturer to construct pLarge.

To construct pLargeRhaLysisE, a synthetic DNA sequence encoding lysisprotein E from bacteriophage phiX174 was obtained from MWG. Thesynthetic DNA fragment possessed EcoRI and BamHI sites at the 5′ and 3′side of the coding sequence, respectively. This allowed cloning into theEcoRI and BamHI sites of pLarge, yielding pLargeRhaLysisE.

TABLE 3 Primers used in Example 20 SEQ ID Name NO  Sequence (5′ à 3′)kanR 124 agaaaaacucatcgagcatcaaatg kanF 125 atgagccauattcaacgggaaacpRhakanF 126 agtttttcuaactgtcagaccaagtttactc pRhakanR 127atggctcauactcttcctttttcaatattattgaagc pSB3398  128atctttcugcgaattgagatgac fw pSB3398  129 aagcctaguctcatgagcgg rev 67kF130 actaggctugtaatcatggtcatagctgtttc pRhaR 131agaaagauagacgaaagggcctcgtgatac NB: In Table 3 uracils are indicated withu.

TABLE 4 Constructs used in Example 20 Protein DNA Name SEQ ID NO SEQ IDNO lysis protein E 132 133

Example 21

The following example 21 relates to construction of Salmonella strains.FIG. 33 shows a schematic representation of the location of hbp mutantinsertions into the Salmonella typhimurium SL3261 chromosome. Table 5below shows the primer sequences used in Example 21.

Salmonella typhimurium strains carrying a single copy of either of thegenes encoding Hbp-Ag85B(C+N)-ESAT6-Rv2660c, HbpD-ESAT6 orHbpD-Ag85B(C+N)-ESAT6-Rv2660c on the chromosome, were constructed asfollows. The concerning hbp mutant genes were inserted into thechromosome of S. typhimurium by allelic exchange through doublecross-over homologues recombination (Kaniga et al 1991 Gene 109:137-141), replacing the malE and malK promotor regions. Briefly, the hbpmutants including the lacUV5 promoter region were amplified by PCR usingpHbp-Ag85B(C+N)-ESAT6-Rv2660c, pHbpD-ESAT6 orpHbpD-Ag85B(C+N)-ESAT6-Rv2660c as a template, respectively. The primersused were lacUV5_ScaI_f and pEH3Hbpbeta_ScaI_r. The PCR products weredigested with ScaI and cloned into a SmaI-cut pSB890-derived suicidevector (Palmer et al 1998 Molecular Microbiology 27: 953-965), just inbetween 1000 bp homology regions to malE and ma/K (FIG. 33).

The resulting hbp mutant-suicide vectors were transformed into the E.coli donor strain SM10λ pir (Miller and Mekalanos 1998 Journal ofBacteriology 170: 2575-2583). SM10λ pirwas mated over night on platewith the S. thypimurium recipient strain SL3261 (Hoiseth and Stocker1981 Nature 291: 281-282). Tetracyclin resistant S. thypimuriumtransconjugants were selected on plate.

Resolution of merodiploids and replacement of the wild-type locus withan hbp mutant gene were achieved by selecting for resistance of theSalmonella mutants to sucrose (Kaniga et al 1991 Gene 109: 137-141).Positive clones were identified by PCR of the intergenic region betweenmalE and malK using primers malE_insert_seq and malK_insert_seq, andsequencing of the introduced allele.

TABLE 5 Primers used in Example 21 SEQ ID Name NO Sequence (5′ à 3′)lacUV5_ScaI_f 134 GCGC AGTACT TTG CGC CAT TCT ATG GTG TCpEH3Hbpbeta_ScaI_r 135 GCGCAGTACTCACAGCATCAGA ATGAATAACG malE_insert_seq136 TAT AAC CCT TGT CGC CGT TG malK_insert_seq 137ACG CAG CAA GGT CGA TTT AC

1. A host cell capable of expressing more than one POI (polypeptide ofinterest), the host cell comprising a fusion protein or a nucleic acidencoding a fusion protein, said fusion protein comprising said POI:s andi. a passenger domain comprising a beta stem domain from anautotransporter protein, wherein the beta stem forming sequence of thepassenger domain is essentially intact; ii. a translocator domain froman autotransporter protein; and iii. a signal peptide being able totarget the fusion protein to the inner membrane of Gram negativebacteria; wherein the passenger domain of the autotransporter in itsnative form comprises at least two side domains, and wherein at leasttwo POI:s are inserted into, replace or partly replace a separate sidedomain.
 2. The host cell of claim 1, wherein the fusion protein, whenexpressed, is displayed at the cell surface.
 3. The host cell of claim1, wherein the fusion protein, when expressed, is secreted and releasedfrom the cell surface.
 4. The host cell of claim 1, wherein thepassenger domain in i) and the translocator domain in ii) is derivedfrom a SPATE (serine protease autotransporters of Enterobacteriaceae)protein.
 5. The host cell of claim 4, wherein the SPATE protein isHemoglobin-binding protease (Hbp), extracellular serine protease (EspC)or temperature-sensitive hemagglutinin (Tsh) from Escherichia coli. 6.The host cell of claim 5, wherein the SPATE protein comprises apolypeptide with a sequence that is at least 90% similar to SEQ ID NO 1or SEQ ID NO
 2. 7. The host cell of claim 6, wherein amino acids 53-308,533-608, 657-697, 735-766 and 898-922 of SEQ ID NO 1 or SEQ ID NO 2correspond to side domains, and wherein the POI:s are inserted into,replace or partly replace at least two of such side domains.
 8. A fusionprotein comprising i. more than one POI (polypeptide of interest) ii. apassenger domain comprising a beta stem domain from an autotransporterprotein, wherein the beta stem forming sequence of the passenger domainis essentially intact; iii. a translocator domain from anautotransporter protein; and iv. optionally, a signal peptide thattargets the fusion protein to the inner membrane of a Gram negativebacterium, wherein the passenger domain of the autotransporter in itsnative form comprises at least two side domains, and wherein at leasttwo POI:s replace or partly replace a separate side domain.
 9. Thefusion protein of claim 8, wherein the passenger domain in i) and thetranslocator domain in ii) is derived from a SPATE (serine proteaseautotransporters of Enterobacteriaceae) protein.
 10. The fusion proteinof claim 9, wherein the SPATE protein is Hemoglobin-binding protease(Hbp), extracellular serine protease (EspC) or temperature-sensitivehemagglutinin (Tsh) from Escherichia coli.
 11. The fusion protein ofclaim 10, wherein the SPATE protein comprises a polypeptide with asequence that is at least 90% similar to SEQ ID NO 1 or SEQ ID NO
 2. 12.The fusion protein of claim 11, wherein amino acids 53-308, 533-608,657-697, 735-766 and 898-922 of SEQ ID NO 1 or SEQ ID NO 2 correspond toside domains, and wherein the POI:s are inserted into, replace or partlyreplace at least two of such side domains.
 13. A nucleic acid arrangedfor expression of a fusion protein, said nucleic acid comprising, inframe: i. sequence encoding a signal peptide of said fusion protein, thesignal peptide being able to target the fusion protein to the innermembrane of Gram negative bacteria; ii. sequence encoding a passengerdomain of said fusion protein, the passenger domain comprising a betastem domain from an autotransporter protein; and iii. sequence encodinga translocator domain of said fusion protein, the translocator domainderiving from an autotransporter protein, wherein the sequence encodingthe passenger domain of the autotransporter in its native form comprisesat least two stretches of sequence encoding side domains protruding fromthe beta stem domain, and wherein the sequence encoding the passengerdomain comprises at least two stretches of cloning site sequence thatallow in-frame cloning of at least two DNA sequences that encode POI:s(polypeptides of interest), said at least two stretches of cloning sitesequence being inserted into, replacing or partly replacing separatestretches of said stretches of sequence encoding side domains and saidstretches of cloning site sequences being arranged such that the encodedbeta stem forming protein sequence of the passenger domain isessentially intact.
 14. A nucleic acid arranged for expression of afusion protein, said nucleic acid comprising, in frame: i. sequenceencoding a signal peptide of said fusion protein, the signal peptidebeing able to target the fusion protein to the inner membrane of Gramnegative bacteria; ii. sequence encoding a passenger domain of saidfusion protein, the passenger domain comprising a beta stem domain froman autotransporter protein; iii. sequence encoding a translocator domainof said fusion protein, the translocator domain deriving from anautotransporter protein; and iv. sequences encoding more than one POI(polypeptide of interest) of said fusion protein, wherein the sequencesencoding the POI:s are fused to the sequence encoding the passengerdomain and are arranged such that the encoded beta stem forming proteinsequence of the passenger domain is essentially intact, and wherein thesequence encoding the passenger domain of the autotransporter in itsnative form comprises at least two stretches of sequence encoding sidedomains protruding from the beta stem domain, and each of the sequencesencoding POI:s are inserted into, replace or partly replace separatestretches of said stretches of sequence encoding side domains.
 15. Thenucleic acid of claim 13, further comprising sequence encoding for acleavage site that allows for secretion of the encoded fusion proteinfrom a host cell harboring said nucleic acid.
 16. The nucleic acid ofclaim 13, comprising no sequence encoding for a cleavage site thatallows for secretion, or comprising a disrupted cleavage site, such thatthe encoded fusion protein is arranged to be displayed on the cellsurface of a host cell harboring said nucleic acid.
 17. The nucleic acidof claim 13, wherein the sequences encoding the passenger domain in ii)and the translocator domain in iii) are derived from a gene encoding aSPATE (serine protease autotransporters of Enterobacteriaceae) protein.18. The nucleic acid of claim 17, wherein the SPATE protein isHemoglobin-binding protease (Hbp), extracellular serine protease (EspC)or temperature-sensitive hemagglutinin (Tsh) from Escherichia coli. 19.The nucleic acid of claim 18, wherein the gene encoding the SPATEprotein encodes a protein sequence that is at least 90% similar to SEQID NO 1 or SEQ ID NO
 2. 20. The nucleic acid of claim 19, wherein aminoacids 53-308, 533-608, 657-697, 735-766 and 898-922 of SEQ ID NO 1 orSEQ ID NO 2 correspond to side domains, and wherein the cloning sites orthe sequences encoding POI:s are arranged to replace or partly replaceat least two of such side domains.
 21. A vector comprising a nucleicacid according to claim
 13. 22. A host cell comprising a nucleic acid orvector according to claim
 13. 23. The host cell according to claim 1,wherein the host cell is a Gram negative bacterium.
 24. The host cellaccording to claim 23, which is selected from the family ofEnterobacteriaceae, such as Escherichia coli, Salmonella spp., Vibriospp., Shigella spp., Pseudomonads spp., Burkholderia spp. or Bordetellaspp.
 25. An outer membrane vesicle displaying a fusion protein accordingto claim 8 on its surface.
 26. A bacterial ghost displaying a fusionprotein according to claim 8 on its surface.
 27. A method for secretoryprotein expression of a fusion protein, comprising the steps of i.Providing a host cell according to claim 1; ii. Inducing expression ofthe fusion protein.
 28. The method of claim 27 comprising the additionalstep of inhibiting a periplasmic enzyme with protease activity in thehost cell.
 29. The method of claim 28, wherein the enzyme is DegP. 30.The method of claim 29, wherein DegP is inhibited by a mutation in thecatalytic site of DegP.
 31. The method of claim 27 comprising theadditional step of down regulating at least one enzyme that catalyzesthe formation of disulphide bonds in proteins in the periplasmic spaceof the host cell.
 32. The method of claim 31, wherein the enzyme is DsbAor DsbB.
 33. The method of claim 27, wherein the fusion protein issecreted in a soluble manner.
 34. The method of claim 27, wherein thefusion protein is displayed on the cell surface.
 35. The method of claim27, wherein the host cell is a Gram negative bacterium and wherein themethod comprises the additional step of inducing shedding of vesiclesfrom the outer membrane of the Gram negative bacterium, thus formingouter membrane vesicles displaying the fusion protein on their surface.36. The method of claim 27, wherein the host cell is a Gram negativebacterium and wherein the method comprises the additional step of lysingthe Gram negative bacterium to form bacterial ghosts displaying thefusion protein on their surface.
 37. The method of claim 36, wherein thelysing is made by use of the lethal lysis gene E from bacteriophagePhiX174.
 38. The host cell of claim 1, wherein at least one of the POI:scomprises an antigen.
 39. The host cell of claim 38, wherein the antigenis an antigen from Mycobacterium tuberculosis.
 40. The host cell ofclaim 39, wherein the antigen from Mycobacterium tuberculosis isselected from the group consisting of ESAT-6, Ag85B, Rv2660c, TB10.4 andTB10.3, or a protein that is similar to those proteins.
 41. The hostcell of claim 40, wherein the antigen is Ag85B that has been split intoa N′-part (Ag85B(N′)) and a C′-part (Ag85B(C′)), and wherein each partis fused to a separate side domain of a passenger from anautotransporter protein.
 42. The host cell of claim 40, wherein at leasttwo of the antigens ESAT-6, Ag85B, Rv2660c, TB10.4 and TB10.3, eacheither split or in full sequence, are fused to, inserted into, replaceor partly replace a separate side domain of a passenger from anautotransporter protein.
 43. A vaccine comprising a host cell accordingto claim
 38. 44. The fusion protein of claim 8, wherein at least one ofthe POI:s comprises an antigen.
 45. The fusion protein of claim 44,wherein the antigen is an antigen from Mycobacterium tuberculosis. 46.The fusion protein of claim 44, wherein the antigen from Mycobacteriumtuberculosis is selected from the group consisting of ESAT-6, Ag85B,Rv2660c, TB10.4 and TB10.3, or a protein that is similar to thoseproteins.
 47. The fusion protein of claim 44, wherein the antigen isAg85B that has been split into a N′-part (Ag85B(N′)) and a C′-part(Ag85B(C′)), and wherein each part is fused to a separate side domain ofa passenger from an autotransporter protein.
 48. The fusion protein ofclaim 44, wherein at least two of the antigens ESAT-6, Ag85B, Rv2660c,TB10.4 and TB10.3, each either split or in full sequence, are fused to,inserted into, replace or partly replace a separate side domain of apassenger from an autotransporter protein.
 49. A vaccine comprising thefusion protein according to claim
 44. 50. The nucleic acid of claim 13,wherein at least one of the POI:s comprises an antigen.
 51. The nucleicacid of claim 50, wherein the antigen is an antigen from Mycobacteriumtuberculosis.
 52. The nucleic acid of claim 50, wherein the antigen fromMycobacterium tuberculosis is selected from the group consisting ofESAT-6, Ag85B, Rv2660c, TB10.4 and TB10.3, or a protein that is similarto those proteins.
 53. The nucleic acid of claim 50, wherein the antigenis Ag85B that has been split into a N′-part (Ag85B(N′)) and a C′-part(Ag85B(C′)), and wherein each part is fused to a separate side domain ofa passenger from an autotransporter protein.
 54. The nucleic acid ofclaim 50, wherein at least two of the antigens ESAT-6, Ag85B, Rv2660c,TB10.4 and TB10.3, each either split or in full sequence, are fused to,inserted into, replace or partly replace a separate side domain of apassenger from an autotransporter protein.
 55. A vaccine comprising thenucleic acid according to claim
 50. 56. The method of claim 27, whereinat least one of the POI:s expressed by the host cell comprises anantigen.
 57. The method of claim 56, wherein the antigen is an antigenfrom Mycobacterium tuberculosis.
 58. The method of claim 56, wherein theantigen from Mycobacterium tuberculosis is selected from the groupconsisting of ESAT-6, Ag85B, Rv2660c, TB10.4 and TB10.3, or a proteinthat is similar to those proteins.
 59. The method of claim 56, whereinthe antigen is Ag85B that has been split into a N′-part (Ag85B(N′)) anda C′-part (Ag85B(C′)), and wherein each part is fused to a separate sidedomain of a passenger from an autotransporter protein.
 60. The method ofclaim 56, wherein at least twoof the antigens ESAT-6, Ag85B, Rv2660c,TB10.4 and TB10.3, each either split or in full sequence, are fused to,inserted into, replace or partly replace a separate side domain of apassenger from an autotransporter protein.
 61. A method for preparing avaccine comprising the method according to claim 56.